U.S. patent application number 13/004158 was filed with the patent office on 2011-07-28 for full length large t tumor antigen of merkel cell polyomavirus as a therapeutic target in merkel cell carcinoma.
This patent application is currently assigned to INSTITUT CURIE. Invention is credited to Martine PETER, Xavier SASTRE-GARAU.
Application Number | 20110182901 13/004158 |
Document ID | / |
Family ID | 44309120 |
Filed Date | 2011-07-28 |
United States Patent
Application |
20110182901 |
Kind Code |
A1 |
PETER; Martine ; et
al. |
July 28, 2011 |
FULL LENGTH LARGE T TUMOR ANTIGEN OF MERKEL CELL POLYOMAVIRUS AS A
THERAPEUTIC TARGET IN MERKEL CELL CARCINOMA
Abstract
The invention relates to a full length Large T tumor antigen of
Merkel Cell Polyomavirus (MCV) as a therapeutic target in Merkel
Cell Carcinoma (MCC).
Inventors: |
PETER; Martine; (Paris,
FR) ; SASTRE-GARAU; Xavier; (Paris, FR) |
Assignee: |
INSTITUT CURIE
Paris Cedex 5
FR
|
Family ID: |
44309120 |
Appl. No.: |
13/004158 |
Filed: |
January 11, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61298343 |
Jan 26, 2010 |
|
|
|
Current U.S.
Class: |
424/139.1 ;
424/186.1; 424/204.1; 435/235.1; 435/5; 436/501; 436/86; 436/94;
514/44R; 530/350; 530/387.9; 530/389.4; 536/23.1; 536/23.72 |
Current CPC
Class: |
Y02A 50/467 20180101;
G01N 33/5743 20130101; C12N 7/00 20130101; C12N 2710/22023
20130101; G01N 2333/01 20130101; Y02A 50/30 20180101; A61P 35/00
20180101; A61P 37/04 20180101; C12N 2710/22022 20130101; C12Q 1/701
20130101; Y10T 436/143333 20150115; A61P 31/20 20180101; C12N
2710/22021 20130101; G01N 2500/02 20130101; C07K 14/005 20130101;
A61K 31/7088 20130101 |
Class at
Publication: |
424/139.1 ;
435/235.1; 536/23.72; 536/23.1; 530/350; 530/389.4; 530/387.9;
435/5; 436/501; 436/86; 436/94; 514/44.R; 424/186.1; 424/204.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 7/00 20060101 C12N007/00; C07H 21/00 20060101
C07H021/00; C07K 14/01 20060101 C07K014/01; C07K 16/08 20060101
C07K016/08; C12Q 1/70 20060101 C12Q001/70; G01N 33/68 20060101
G01N033/68; G01N 33/53 20060101 G01N033/53; A61K 31/7088 20060101
A61K031/7088; A61K 39/12 20060101 A61K039/12; A61P 31/20 20060101
A61P031/20; A61P 35/00 20060101 A61P035/00; A61P 37/04 20060101
A61P037/04 |
Claims
1. A virus-like particle (VLP) wherein said VLP is a polyomavirus
comprising a nucleic acid sequence having at least 60% identity
with SEQ ID NO:5 (exon 2 of large T antigen, LT) and wherein said
VLP has been isolated from a patient, preferably a patient
suffering from Merkel Cell Carcinoma (MCC) in an episomal form or
integrated in the patient's genome.
2. A VLP according to claim 1 comprising a polypeptide of at least
470 amino acids having at least 60% identity with SEQ ID NO:6 (LT)
over said at least 470 amino acids.
3. A VLP according to claim 2 comprising a polypeptide having at
least 60% identity with SEQ ID NO:6.
4. A VLP according to claim 1, further comprising at least one
nucleic acid selected from the group consisting of the nucleic
acids having: at least 99.4% identity with SEQ ID NO: 5 (exon 2 of
LT); at least 99.2% identity with SEQ ID NO:4 (exon 1 of LT); at
least 99.5% identity with SEQ ID NO:2 (ST); at least 99.5% identity
with SEQ ID NO:7 (VP1); at least 99.5% identity with SEQ ID NO:9
(VP2); at least 99.5% identity with SEQ ID NO:11 (VP3) and at least
99.4% identity with SEQ ID NO:1 (full genome of MCV-IC13).
5. An isolated nucleic acid selected from the group consisting of a
nucleic acid having at least 99.4% identity with SEQ ID NO:1, a
nucleic acid having at least 99.5% identity with SEQ ID NO:2, a
nucleic acid having at least 99.2% identity with SEQ ID NO:4, a
nucleic acid having at least 99.4% identity with SEQ ID NO:5, a
nucleic acid having at least 99.5% identity with SEQ ID NO:7, a
nucleic acid having at least 99.5% identity with SEQ ID NO:9 and a
nucleic acid having at least 99.5% identity with SEQ ID NO:11.
6. A isolated polypeptide selected from the group consisting of an
amino acid sequence having at least 99.6% identity with SEQ ID
NO:3, an amino acid sequence having at least 98.6% identity with
SEQ ID NO:6, an amino acid sequence having at least 99.4% identity
with SEQ ID NO:8, an amino acid sequence having at least 99.3%
identity with SEQ ID NO:10 and an amino acid sequence having at
least 99.0% identity with SEQ ID NO:12, or a fragment of said
polypeptide having at least 99.6% identity with the corresponding
fragments of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10
and SEQ ID NO:12, respectively.
7. An anti-MCV agent wherein said anti-MCV agent is a molecule
which specifically interacts with a nucleic acid according to claim
5.
8. An anti-MCV agent wherein said anti-MCV agent inhibits the
expression and/or the activity of at least one nucleic acid
according to claim 5.
9. An antibody which specifically recognizes the non-truncated LT
protein.
10. An antibody according to claim 9, wherein said antibody
specifically recognizes an antigen comprised between amino acids
456 to 817 of SEQ ID NO: 6.
11. An antibody according to claim 9, wherein said antibody
specifically recognizes a conformational epitope wherein said
conformational epitope is partly comprised of residues located
between amino acids 456 to 817 of SEQ ID NO: 6.
12. A method for detecting a VLP according to claim 1, comprising
the step of detecting a nucleic acid selected from the group
consisting of a nucleic acid having at least 99.4% identity with
SEQ ID NO:1, a nucleic acid having at least 99.5% identity with SEQ
ID NO:2, a nucleic acid having at least 99.2% identity with SEQ ID
NO:4, a nucleic acid having at least 99.4% identity with SEQ ID
NO:5, a nucleic acid having at least 99.5% identity with SEQ ID
NO:7, a nucleic acid having at least 99.5% identity with SEQ ID
NO:9 and a nucleic acid having at least 99.5% identity with SEQ ID
NO:11; or detecting a polypeptide selected from the group
consisting of an amino acid sequence having at least 99.6% identity
with SEQ ID NO:3, an amino acid sequence having at least 98.6%
identity with SEQ ID NO:6, an amino acid sequence having at least
99.4% identity with SEQ ID NO:8, an amino acid sequence having at
least 99.3% identity with SEQ ID NO:10 and an amino acid sequence
having at least 99.0% identity with SEQ ID NO:12, or a fragment of
said polypeptide having at least 99.6% identity with the
corresponding fragments of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8,
SEQ ID NO:10 and SEQ ID NO:12, respectively.
13. A method according to claim 12, comprising the step of
detecting an LT protein with an antibody which specifically
recognizes the non-truncated LT protein.
14. A method for predicting the risk of developing an
MCV-associated disease in a patient comprising the step of
detecting a VLP according to claim 1 in a tissue sample obtained
from said patient.
15. A method for diagnosing an MCV-associated disease in a patient
comprising the step of detecting a VLP according to claim 1 in a
tissue sample obtained from said patient.
16. A method according to claim 14, wherein said MCV-associated
disease is Merkel Cell Carcinoma (MCC).
17. A kit for diagnosis an MCV-associated disease in a patient
comprising an anti-MCV agent according to claim 7 and means for
revealing said anti-MCV agent or antibody.
18. A method for identifying an agent that attenuates MCV infection
comprising the step of exposing a target DNA to a polypeptide
according to claim 6 in the presence or absence of a test
compound.
19. A pharmaceutical composition comprising: a VLP according to
and/or a nucleic acid selected from the group consisting of a
nucleic acid having at least 99.4% identity with SEQ ID NO:1, a
nucleic acid having at least 99.5% identity with SEQ ID NO:2, a
nucleic acid having at least 99.2% identity with SEQ ID NO:4, a
nucleic acid having at least 99.4% identity with SEQ ID NO:5, a
nucleic acid having at least 99.5% identity with SEQ ID NO:7, a
nucleic acid having at least 99.5% identity with SEQ ID NO:9 and a
nucleic acid having at least 99.5% identity with SEQ ID NO:11
and/or a polypeptide selected from the group consisting of an amino
acid sequence having at least 99.6% identity with SEQ ID NO:3, an
amino acid sequence having at least 98.6% identity with SEQ ID
NO:6, an amino acid sequence having at least 99.4% identity with
SEQ ID NO:8, an amino acid sequence having at least 99.3% identity
with SEQ ID NO:10 and an amino acid sequence having at least 99.0%
identity with SEQ ID NO:12, or a fragment of said polypeptide
having at least 99.6% identity with the corresponding fragments of
SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID
NO:12, respectively and/or an anti-MCV agent which is a molecule
which specifically interacts with a nucleic acid selected from the
group consisting of a nucleic acid having at least 99.4% identity
with SEQ ID NO:1, a nucleic acid having at least 99.5% identity
with SEQ ID NO:2, a nucleic acid having at least 99.2% identity
with SEQ ID NO:4, a nucleic acid having at least 99.4% identity
with SEQ ID NO:5, a nucleic acid having at least 99.5% identity
with SEQ ID NO:7, a nucleic acid having at least 99.5% identity
with SEQ ID NO:9 and a nucleic acid having at least 99.5% identity
with SEQ ID NO:11, or specifically interacts with a polypeptide
selected from the group consisting of an amino acid sequence having
at least 99.6% identity with SEQ ID NO:3, an amino acid sequence
having at least 98.6% identity with SEQ ID NO:6, an amino acid
sequence having at least 99.4% identity with SEQ ID NO:8, an amino
acid sequence having at least 99.3% identity with SEQ ID NO:10 and
an amino acid sequence having at least 99.0% identity with SEQ ID
NO:12, or a fragment of said polypeptide having at least 99.6%
identity with the corresponding fragments of SEQ ID NO:3, SEQ ID
NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12, respectively and
a pharmaceutically acceptable carrier.
Description
FIELD OF THE INVENTION
[0001] The invention relates to a full length Large T tumor antigen
of Merkel Cell Polyomavirus as a therapeutic target in Merkel Cell
Carcinoma.
BACKGROUND OF THE INVENTION
[0002] Merkel cell carcinoma (MCC) is a cutaneous tumour [1] with
neuroendocrine features and poor outcome [2-4]. This tumour
develops in the sun-exposed areas of the skin in elderly or
immunosuppressed individuals [5]. A three-fold increase in
incidence has been observed in the United States over the past 15
years [6], attributable in part to an aging population with
extensive sun exposure. Although classically believed to be derived
from Merkel neuroendocrine epidermal cells [2], the histogenesis of
this tumour is still under debate [3]. Studies on the molecular
origins of MCC thus far have provided only negative results [7] and
this lack of knowledge limits the development of targeted
therapies.
[0003] Polyomaviruses are small non enveloped viruses with a double
stranded circular DNA chromosome composed of 4700 to 5400 bp. This
genome encodes the three structural proteins which constitute the
viral particle (VP1, 2, 3) and two early tumour antigens, called
small T (ST) and large T (LT). In their natural host,
polyomaviruses are non-oncogenic: infection leads to the
replication of the viral genome and production of viral particles
resulting in cell lysis. In contrast, in heterologous
experimentally transformed cells, no production of infectious virus
occurs. The non permissive host cells integrate into their genome
viral DNA sequences which constitutively express ST and LT
antigens.
[0004] A new type of human polyomavirus was recently identified in
MCC, thus called Merkel Cell Polyomavirus (MCV or MCPyV) [8]. The
presence of MCV DNA sequences in MCC has been confirmed in three
series of cases [9-11]. This association does not however establish
a causal role for MCV in MCC.
[0005] In particular, these authors have identified and sequenced
two clones of MCV: MCV350 and MCV339. The complete sequences of
MCV350 and MCV339 are available under Genbank accession numbers
EU375803 and EU375804, respectively.
[0006] Document WO 2009/079481 describes isolated or substantially
purified polypeptides, nucleic acids, and virus-like particles
(VLPs) derived from said clones. A common feature of MCV350 and
MCV339 and other clones of MCV found in association with cancerous
tissues is that the sequence encoding the Large T antigen (LT)
results in a truncated protein. The authors speculate that tumours
strongly select against retention of intact MCV large antigen.
[0007] Surprisingly, the inventors have identified a novel clone of
MCV associated with MCC, named MCV IC-13, which contains a full
length LT antigen.
SUMMARY OF THE INVENTION
[0008] The inventors have identified a novel clone of Merkel Cell
Polyomavirus (MCV) associated with MCC, whose genome presents
several differences compared to the known clones of MCV and in
particular which encodes a full length LT antigen.
[0009] Thus, in one aspect, the invention relates to a virus-like
particle (VLP) wherein said VLP is derived from a polyomavirus,
preferably a Merkel Cell Polyomavirus (MCV) comprising a nucleic
acid sequence having at least 60% identity with SEQ ID NO:5 (exon 2
of large T antigen, LT) and wherein said VLP has been isolated from
a patient, preferably a patient suffering from Merkel Cell
Carcinoma (MCC) in an episomal form or integrated in the patient's
genome.
[0010] In another aspect, the invention also relates to an isolated
nucleic acid selected from the group consisting of a nucleic acid
having at least 99.4% identity with SEQ ID NO:1, a nucleic acid
having at least 99.5% identity with SEQ ID NO:2, a nucleic acid
having at least 99.2% identity with SEQ ID NO:4, a nucleic acid
having at least 99.4% identity with SEQ ID NO:5, a nucleic acid
having at least 99.5% identity with SEQ ID NO:7, a nucleic acid
having at least 99.5% identity with SEQ ID NO:9 and a nucleic acid
having at least 99.5% identity with SEQ ID NO:11.
[0011] In another aspect, the invention relates to a isolated
polypeptide selected from the group consisting of an amino acid
sequence having at least 99.6% identity with SEQ ID NO:3, an amino
acid sequence having at least 98.6% identity with SEQ ID NO:6, an
amino acid sequence having at least 99.4% identity with SEQ ID
NO:8, an amino acid sequence having at least 99.3% identity with
SEQ ID NO:10 and an amino acid sequence having at least 99.0%
identity with SEQ ID NO:12, or a fragment of said polypeptide
having at least 99.6% identity with the corresponding fragments of
SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID
NO:12, respectively.
[0012] In yet another aspect, the invention relates to an anti-MCV
agent wherein said anti-MCV agent is a molecule which specifically
interacts with a nucleic acid or a polypeptide as described
above.
[0013] In particular, the invention pertains to an antibody which
specifically recognizes an antigen comprised between amino acids
456 to 817 of SEQ ID NO: 6.
[0014] The invention also relates to methods and kits for detecting
a VLP as defined above and to methods and kits for predicting the
risk of developing an MCV-associated disease in a patient or for
diagnosing an MCV-associated disease in a patient comprising the
step of detecting a VLP as defined above in a tissue sample
obtained from said patient.
[0015] The invention also relates to a method for identifying an
agent that attenuates MCV infection comprising the step of exposing
a target DNA to a polypeptide as defined above in the presence or
absence of a test compound.
[0016] The invention also relates to a pharmaceutical composition
comprising one or several of the elements as defined above and a
pharmaceutically acceptable carrier.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The inventors have identified a novel clone of Merkel Cell
Polyomavirus (MCV) associated with Merkel cell Carcinoma (MCC),
which presents several differences compared to the known clones of
MCV. This clone is identified as MCV-IC13.
[0018] The genome of said MCV IC-13 consists in the nucleic acid
sequence as set forth in SEQ ID NO:1.
[0019] The overall identity between MCV IC-13 and the MCV clones of
the prior art is 99.35% with MCV350 and 96% with MCV339. However,
one principal difference between said clones resides in the fact
that the clone of the MCV possesses a full length LT antigen.
[0020] The genome of MCV IC-13 contains 5 genes (see Table 1):
[0021] the nucleic acid sequence spanning from positions 196 to 756
of SEQ ID NO:1 (SEQ ID NO:2), which encodes the small T antigen
(ST) having the amino acid sequence as set forth in SEQ ID NO:3;
[0022] the nucleic acid sequences spanning from positions 196 to
429 of SEQ ID NO:1 (SEQ ID NO:4) and from positions 861 to 3080 of
SEQ ID NO:1 (SEQ ID NO:5), which correspond to exons 1 and 2
respectively of the large T antigen (LT). [0023] The amino acid
sequence of LT is the sequence as set forth in SEQ ID NO: 6; [0024]
the nucleic acid sequences spanning from positions 3156 to 4427 of
SEQ ID NO:1 (SEQ ID NO:7), from positions 4393 to 5118 of SEQ ID
NO: 1 (SEQ ID NO:9) and from positions 4393 to 4983 of SEQ ID NO:1
(SEQ ID NO:11), encoding the three structural viral proteins VP1,
VP2 and VP3, respectively. [0025] The amino acid sequences of VP1,
VP2 and VP3 are respectively set forth as SEQ ID NO: 8, SEQ ID NO:
10 and SEQ ID NO: 12.
[0026] As can be seen in Table 1, the MCV clone identified by the
inventors, named MCV-IC13, presents several differences with the
previously identified clones, MCV 350 and MCV 339. In particular:
[0027] the LT protein as set forth in SEQ ID NO:6 is a full length
protein of 817 amino acids, whereas that of MCV350 contains a stop
codon at position 259 and that of MCV339 contains a deletion which
also results in a truncated protein of 469 amino acids; [0028] the
sequence of the ST protein as set forth in SEQ ID NO:3 differs by
one amino acid from those of MCV350 and MCV339; [0029] the
sequences of VP1, VP2 and VP3 viral proteins (SEQ ID NO:8, SEQ ID
NO:10 and SEQ ID NO:12, respectively) contains several point
mutations compared to their homologues in MCV350 and MCV339;
TABLE-US-00001 [0029] TABLE 1 Comparison of the MCV-IC13 clone with
clones MCV350 and MCV339 Sequence of Nucleic acid Protein MCV-IC13
SEQ ID % identity with MCV % identity with SEQ ID % identity with
MCV % identity with MCV clone NO: positions 350 MCV 339 NO: 350 339
Complete NO: 1 1-5387 99.35% 96% N/A N/A genome ST NO: 2 196-756 nd
nd NO: 3 99.5% 99.5% LT NO: 6 98.5% N/A exon 1 NO: 4 196-429 98.7%
99.10% exon 2 NO: 5 861-3080 99.3% 91.10% VP1 NO: 7 3156-4427 nd nd
NO: 8 98.6% 99.3% VP2 NO: 9 4393-5118 nd nd NO: 10 98.8% 99.2% VP3
NO: 11 4393-4983 nd nd NO: 12 98.9% 98.9% nd: not determined N/A:
not applicable (the percentage could not be calculated, due to the
presence of several proteins encoded by SEQ ID NO: 1 or due to a
large deletion, which would bias the calculation in the case of LT
of MCV 339)
[0030] To determine the percent identity of two nucleic acid
sequences, the sequences are aligned for optimal comparison. For
example, gaps can be introduced in the sequence of a first nucleic
acid sequence for optimal alignment with the second nucleic acid
sequence. The nucleotides at corresponding nucleotide positions are
then compared. When a position in the first sequence is occupied by
the same nucleotide as at the corresponding position in the second
sequence, the nucleic acids are identical at that position. The
percent identity between the two sequences is a function of the
number of identical nucleotides shared by the sequences.
[0031] Hence % identity=[number of identical nucleotides/total
number of overlapping positions].times.100. The percentage of
sequence identity is thus calculated according to this formula, by
comparing two optimally aligned sequences over the window of
comparison, determining the number of positions at which the
identical nucleic acid base (e.g., A, T, C, G) occurs in both
sequences to yield the number of matched positions (the "number of
identical positions" in the formula above), dividing the number of
matched positions by the total number of positions in the window of
comparison (e.g. the window size) (the "total number of overlapping
positions" in the formula above), and multiplying the result by 100
to yield the percentage of sequence identity.
[0032] In this comparison, the sequences can be the same length or
may be different in length. Optimal alignment of sequences for
determining a comparison window may be conducted by the local
identity algorithm of Smith and Waterman (1981), by the identity
alignment algorithm of Needleman and Wunsh (1972), by the search
for similarity via the method of Pearson and Lipman (1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA and TFASTA in the Wisconsin Genetics Software Package Release
7.0, Genetic Computer Group, 575, Science Drive, Madison, Wis.), or
by inspection.
[0033] Without wishing to be bound by theory, it is believed that
the differences in the above sequences result in the
physiopathology of clone MCV IC-13 being different from that of any
known MCV clone. In particular it is believed that, in MCV IC-13,
Small T (ST) and Large T antigens have different biological
properties from those of the prior art. Of particular interest, the
LT antigen of MCV IC-13 is unique in that it possesses a conserved
helicase domain (see FIG. 1).
Viral-Like Particles (VLP) of the Invention
[0034] In one embodiment, the invention relates to a virus-like
particle (VLP) wherein said VLP is derived from a polyomavirus,
preferably a Merkel cell Polyomavirus (MCV), comprising a nucleic
acid sequence having at least 60% identity with SEQ ID NO:5 (exon 2
of large T antigen, LT) and wherein said VLP has been isolated from
a patient, preferably a patient suffering from Merkel Cell
Carcinoma (MCC) in an episomal form or integrated in the patient's
genome.
[0035] In a preferred embodiment, said VLP comprises a nucleic acid
sequence having at least 65% identity with SEQ ID NO:5, preferably
at least 70%, at least 75%, at least 80%, at least 85%, at least
86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% identity
with SEQ ID NO:5, even more preferably at least 99.5%, at least
99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least
99.95% identity with SEQ ID NO:5.
[0036] As used herein, the expression "viral-like particle" or
"VLP" encompasses both viral particles and particles that resemble
the virus from which they were derived but lack viral nucleic acid.
VLP according to the invention therefore comprise at least a viral
envelope. VLP can therefore be infectious (when they comprise viral
nucleic acid) or non-infectious particles (when they are devoid of
nucleic acid).
[0037] As used herein, the term "polyomavirus" has its general
meaning in the art. Polyomaviruses are DNA-based (double-stranded
DNA, .about.5000 base pairs, circular genome), small (40-50
nanometers in diameter), and icosahedral in shape, and do not have
a lipoprotein envelope. They are potentially oncogenic
(tumor-causing); they often persist as latent infections in a host
without causing disease, but may produce tumors in a host of a
different species, or a host with an ineffective immune system. The
name polyoma refers to the viruses' ability to produce multiple
(poly-) tumors (-oma).
[0038] As used herein, the expression "Merkel Cell Polyomavirus" or
"MCV" has its general meaning in the art. MCV is a human
polyomavirus, first discovered in 2008, which is highly divergent
from the other human polyomaviruses and is most closely related to
murine polyomavirus.
[0039] As used herein, the term "patient" can include human
patients as well as animals. In this respect, the diagnostic and
therapeutic methods can be performed in the veterinary context,
i.e., on domestic animals, particularly mammals (e.g., dogs, cats,
etc.) or agriculturally-important animals (e.g., horses, cows,
sheep, goats, etc.) or animals of zoological importance (apes, such
as gorillas, chimpanzees, and orangutans, large cats, such as
lions, tigers, panthers, etc., antelopes, gazelles, and others). In
a preferred embodiment, said patient is a mammalian, preferably a
primate, even more preferably a human patient.
[0040] The VLP of the invention can be isolated from a patient. In
other terms, the VLP according to the invention can be isolated
from a tissue sample of a patient. Within said tissue sample, the
VLP can be present either in an episomal form or integrated in the
patient's genome.
[0041] The skilled person in the art knows how to isolate a VLP
from a patient and can readily determine whether said VLP is
present as an episome, i.e. as a separate nucleic acid molecule, or
whether it is integrated into one of the patient's chromosomes,
using standard techniques in the art.
[0042] In a preferred embodiment, said VLP is isolated from a
patient suffering from Merkel Cell Carcinoma.
[0043] In one embodiment, the VLP of the invention further
comprises a polypeptide of at least 470 amino acids having at least
60% identity with SEQ ID NO:6 (LT polypeptide) over said at least
470 amino acids.
[0044] As used herein, the terms "polypeptide" or "protein" can be
used interchangeably and have their general meaning in the art.
Polypeptides or proteins comprise two or more amino acid residues
linked together by a peptide bond.
[0045] In a preferred embodiment, said polypeptide has at least
70%, preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% identity with SEQ ID NO: 6 over the length of
said polypeptide, even more preferably at least 99.5%, at least
99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least
99.95% identity with SEQ ID NO:6 over the length of said
polypeptide.
[0046] In a preferred embodiment, said polypeptide comprises at
least 500 amino acids, even more preferably at least 600, 700, 750,
760, 770, 780, 790, 800, 810, 811, 812, 813, 814, 815, 816 or 817
amino acids.
[0047] Thus, in one embodiment, the VLP comprises a polypeptide of
about 817 amino acids having at least 60% identity with SEQ ID
NO:6, preferably at least 70%, preferably at least 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID
NO: 6 over the length of said polypeptide, even more preferably at
least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at
least 99.9%, at least 99.95% identity with SEQ ID NO:6.
[0048] In one embodiment, the VLP according to the invention
further comprises at least one nucleic acid selected from the group
consisting of the nucleic acids having: [0049] at least 99.4%
identity with SEQ ID NO: 5 (exon 2 of LT); [0050] at least 99.2%
identity with SEQ ID NO:4 (exon 1 of LT); [0051] at least 99.5%
identity with SEQ ID NO:2 (ST); [0052] at least 99.5% identity with
SEQ ID NO:7 (VP1); [0053] at least 99.5% identity with SEQ ID NO:9
(VP2); [0054] at least 99.5% identity with SEQ ID NO:11 (VP3) and
[0055] at least 99.4% identity with SEQ ID NO:1 (complete genome of
MCV-IC13).
[0056] In one embodiment, the VLP according to the invention
comprises a nucleic acid having at least 99.4% identity with the
sequence as set forth in SEQ ID NO:5 (exon 2 of LT).
[0057] In a preferred embodiment, said sequence has at least 99.5%
identity with the sequence as set forth in SEQ ID NO:5, even more
preferably at least 99.6%, 99.7%, 99.8%, 99.9% or 99.95% identity
with the sequence as set forth in SEQ ID NO: 5.
[0058] In one embodiment, the VLP according to the invention
comprises a nucleic acid having at least 99.2% identity with the
sequence as set forth in SEQ ID NO: 4 (exon 1 of LT).
[0059] In a preferred embodiment, said sequence has at least 99.3%
identity with the sequence as set forth in SEQ ID NO:4, even more
preferably at least 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or
99.95% identity with the sequence as set forth in SEQ ID NO: 4.
[0060] In one embodiment, the VLP according to the invention
comprises a nucleic acid having at least 99.4% identity with the
sequence as set forth in SEQ ID NO: 1 (complete genome of
MCV-IC13).
[0061] In a preferred embodiment, said nucleic acid has at least
99.5% identity with the sequence as set forth in SEQ ID NO: 1, even
more preferably 99.6%; 99.7%; 99.8%; 99.9% or 99.95% identity with
the sequence as set forth in SEQ ID NO: 1.
[0062] Typically said isolated virus is not selected from the group
consisting of MCV350, MCV339, MCV352 and MCV MKL1.
[0063] In one embodiment, the genome of said isolated virus
consists essentially in SEQ ID NO:1.
[0064] As used herein, the expression "consists essentially in"
means that, out of a nucleic acid sequence of several thousands of
base pairs, minor differences in sequence are tolerated, so long as
they do not impede in the function of the nucleic acid.
Nucleic Acids and Polypeptides of the Invention
[0065] The invention also relates to an isolated nucleic acid
selected from the group consisting of a nucleic acid having at
least 99.4% identity with SEQ ID NO:1, a nucleic acid having at
least 99.5% identity with SEQ ID NO:2, a nucleic acid having at
least 99.2% identity with SEQ ID NO:4, a nucleic acid having at
least 99.4% identity with SEQ ID NO:5, a nucleic acid having at
least 99.5% identity with SEQ ID NO:7, a nucleic acid having at
least 99.5% identity with SEQ ID NO:9 and a nucleic acid having at
least 99.5% identity with SEQ ID NO:11.
[0066] In one embodiment, the invention relates to a nucleic acid
having at least 99.4% identity with SEQ ID NO: 1.
[0067] In a preferred embodiment, said sequence has at least 99.5%
identity with the sequence as set forth in SEQ ID NO: 1, even more
preferably 99.6%; 99.7%; 99.8%; 99.9% or 99.95% identity with the
sequence as set forth in SEQ ID NO: 1.
[0068] In one embodiment, the invention relates to a nucleic acid
having at least 99.2% identity with the sequence as set forth in
SEQ ID NO: 4 (exon 1 of LT).
[0069] In a preferred embodiment, said sequence has at least 99.3%
identity with the sequence as set forth in SEQ ID NO:4, even more
preferably at least 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or
99.95% identity with the sequence as set forth in SEQ ID NO: 4.
[0070] In one embodiment, the invention relates to a nucleic acid
having at least 99.4% identity with the sequence as set forth in
SEQ ID NO:5 (exon 2 of LT).
[0071] In a preferred embodiment, said sequence has at least 99.5%
identity with the sequence as set forth in SEQ ID NO:5, even more
preferably at least 99.6%, 99.7%, 99.8%, 99.9% or 99.95% identity
with the sequence as set forth in SEQ ID NO: 5.
[0072] The invention also relates to a isolated polypeptide
selected from the group consisting of an amino acid sequence having
at least 99.6% identity with SEQ ID NO:3, an amino acid sequence
having at least 98.6% identity with SEQ ID NO:6, an amino acid
sequence having at least 99.4% identity with SEQ ID NO:8, an amino
acid sequence having at least 99.3% identity with SEQ ID NO:10 and
an amino acid sequence having at least 99.0% identity with SEQ ID
NO:12, or a fragment of said polypeptide having at least 99.6%
identity with the corresponding fragments of SEQ ID NO:3, SEQ ID
NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12, respectively.
[0073] Typically, said fragments are significantly different from
the homologous fragments in MCV 350 and MCV 339.
[0074] In a preferred embodiment, the invention relates to a
fragment of polypeptide having at least 98.6% identity with SEQ ID
NO:6, wherein said fragment encompasses the amino acids 456 to 817
of SEQ ID NO:6. Amino acids 456 to 817 of SEQ ID NO:6 are absent
from both MCV350 and MCV339 due to truncation of the LT protein.
This fragment comprises the helicase domain of the LT antigen.
Anti-MCV Agents of the Invention
[0075] Also provided herein are anti-MCV agents. As used herein,
the expression "anti-MCV agent" refers to a molecule that can be
used to recognize a VLP according to the invention. In particular,
said anti-MCV agents are useful for discriminating between the
MCV-IC13 clone of the invention (and VLP according to the invention
derived therefrom) and the MCV clones of the prior art.
[0076] In a preferred embodiment, said anti-MCV agent is an agent
which can further be used to attenuate an MCV infection.
[0077] The invention therefore relates to an anti-MCV agent wherein
said anti-MCV agent is a molecule which specifically interacts with
a nucleic acid or a polypeptide as described above.
[0078] In one embodiment, detection of a nucleic acid is carried
out by hybridization under stringent conditions with a probe that
is selectively hybridizable with of SEQ ID NO:1.
[0079] Within the context of the present invention, a nucleic acid
sequence is considered to be "selectively hybridizable" to a
reference nucleic acid sequence if the two sequences specifically
hybridize to one another under moderate to high stringency
hybridization and wash conditions. Hybridization conditions are
based on the melting temperature (Tm) of the nucleic acid binding
complex or probe. For example, "maximum stringency" typically
occurs at about Tm-5.degree. C. (5.degree. C. below the Tm of the
probe); "high stringency" at about 5-10.degree. C. below the Tm;
"intermediate stringency" at about 10-20.degree. C. below the Tm of
the probe; and "low stringency" at about 20-25.degree. C. below the
Tm. Functionally, maximum stringency conditions may be used to
identify sequences having strict identity or near-strict identity
with the hybridization probe; while high stringency conditions are
used to identify sequences having about 80% or more sequence
identity with the probe. This is especially true for
polynucleotides having a minimum of from about 18-22 nucleic acids,
but those of ordinary skill in the art are also able to apply these
principals to larger or smaller polynucleotides.
[0080] Moderate and high stringency hybridization conditions are
well known in the art (see, for example, Sambrook et al. Molecular
Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor
Press, Plainview, N.Y., 1989, especially chapters 9 and 11; and
Ausubel F M et al. Current Protocols in Molecular Biology, John
Wiley & Sons, New York, N.Y., 1993). An example of high
stringency conditions includes hybridization at about 42.degree. C.
in 50% formamide, 5.times.SSC, 5.times.Denhardt's solution, 0.5%
SDS and 100 .mu.g/ml denatured carrier DNA followed by washing two
times in 2.times.SSC and 0.5% SDS at room temperature and two
additional times in 0.1.times.SSC and 0.5% SDS at 42.degree. C.
[0081] In a preferred embodiment, said probe has at least 99.4%
identity with SEQ ID NO:5 (exon 5 of LT).
[0082] The invention also relates to an anti-agent which
specifically recognizes a polypeptide as defined above.
[0083] The molecules which specifically interact with a polypeptide
of the invention may be an antibody that may be polyclonal or
monoclonal, preferably monoclonal. In another embodiment, the
molecule which specifically interacts with a polypeptide of the
invention may be an aptamer.
[0084] Polyclonal antibodies of the invention can be raised
according to known methods by administering the appropriate antigen
or epitope to a host animal selected, e.g., from pigs, cows,
horses, rabbits, goats, sheep, and mice, among others. Various
adjuvants known in the art can be used to enhance antibody
production. Although antibodies useful in practicing the invention
can be polyclonal, monoclonal antibodies are preferred.
[0085] Monoclonal antibodies of the invention can be prepared and
isolated using any technique that provides for the production of
antibody molecules by continuous cell lines in culture. Techniques
for production and isolation include but are not limited to the
hybridoma technique originally described by Kohler and Milstein
(1975); the human B-cell hybridoma technique (Cote et al., 1983);
and the EBV-hybridoma technique (Cole et al. 1985). Alternatively,
techniques described for the production of single chain antibodies
(see e.g. U.S. Pat. No. 4,946,778) can be adapted to produce single
chain antibodies. Antibodies useful in practicing the present
invention also include fragments including but not limited to
F(ab')2 fragments, which can be generated by pepsin digestion of an
intact antibody molecule, and Fab fragments, which can be generated
by reducing the disulfide bridges of the F(ab')2 fragments.
Alternatively, Fab and/or scFv expression libraries can be
constructed to allow rapid identification of fragments having the
desired specificity. For example, phage display of antibodies may
be used. In such a method, single-chain Fv (scFv) or Fab fragments
are expressed on the surface of a suitable bacteriophage, e.g.,
M13. Briefly, spleen cells of a suitable host, e.g., mouse, that
has been immunized with a protein are removed. The coding regions
of the VL and VH chains are obtained from those cells that are
producing the desired antibody against the protein. These coding
regions are then fused to a terminus of a phage sequence. Once the
phage is inserted into a suitable carrier, e.g., bacteria, the
phage displays the antibody fragment. Phage display of antibodies
may also be provided by combinatorial methods known to those
skilled in the art. Antibody fragments displayed by a phage may
then be used as part of an immunoassay.
[0086] Aptamers are a class of molecule that represents an
alternative to antibodies in term of molecular recognition.
Aptamers are oligonucleotide or oligopeptide sequences with the
capacity to recognize virtually any class of target molecules with
high affinity and specificity. Such ligands may be isolated through
Systematic Evolution of Ligands by EXponential enrichment (SELEX)
of a random sequence library, as described in Tuerk C. and Gold L.,
1990. The random sequence library is obtainable by combinatorial
chemical synthesis of DNA. In this library, each member is a linear
oligomer, eventually chemically modified, of a unique sequence.
Possible modifications, uses and advantages of this class of
molecules have been reviewed in Jayasena S. D., 1999. Peptide
aptamers consist of conformationally constrained antibody variable
regions displayed by a platform protein, such as E. coli
Thioredoxin A, that are selected from combinatorial libraries by
two hybrid methods (Colas et al., 1996).
[0087] In one aspect, the invention relates to an antibody which
specifically recognizes the non-truncated LT protein. As used
herein, the expression "specifically recognizes the non-truncated
LT protein" means that said antibody binds to a non-truncated LT
protein but does not bind to (or binds with a significantly lower
affinity, i.e. an affinity lower by a factor of at least 5,
preferably by a factor of at least 10, even more preferably by a
factor of at least 100) to the truncated LT proteins of MCV clones
of the prior art. Typically, the antibody of the invention binds to
the LT protein of clone MCV-IC13, but not to the LT protein of
clones MCV350 or MCV339.
[0088] In one aspect, the invention also relates to an antibody
which specifically recognizes the LT protein as set forth in SEQ ID
NO:6 and fragments thereof, wherein said fragments comprises at
least 8 successive amino acids among amino acids 456 to 817 of SEQ
ID NO: 6.
[0089] In a particular embodiment, the invention relates to an
antibody which specifically recognizes an antigen comprised between
amino acids 456 to 817 of SEQ ID NO: 6.
[0090] In another embodiment, the invention relates to an antibody
which specifically recognizes a conformational epitope wherein said
conformational epitope is partly comprised of residues located
between amino acids 456 to 817 of SEQ ID NO: 6. In this embodiment,
the conformational epitope may comprise other residues located
elsewhere in the LT protein, but which are in proximity with said
residues located between amino acids 456 to 817 of SEQ ID NO: 6 in
the 3D structure of the LT protein.
[0091] In one embodiment, said anti-MCV agent wherein said anti-MCV
agent inhibits the expression and/or the activity of at least one
nucleic acid of the invention or at least one polypeptide of the
invention.
Detection Methods and Kits According to the Invention
[0092] The invention also relates to methods and kits for detecting
a VLP as defined above and to methods and kits for predicting the
risk of developing an MCV-associated disease in a patient or for
diagnosing an MCV-associated disease in a patient comprising the
step of detecting a VLP as defined above in a tissue sample
obtained from said patient.
[0093] The anti-MCV agents described above are useful for the
following detection methods.
[0094] In one aspect, the invention relates to a method for
detecting a VLP according to the invention, comprising the step of
detecting a nucleic acid or a polypeptide as defined above.
[0095] In a preferred embodiment, the invention relates to a method
for detecting a VLP according to the invention, comprising the step
of detecting an LT protein with an antibody that specifically
recognizes full length LT. Said antibody can be for instance and
antibody directed against an epitope comprised between amino acids
456 to 817 of SEQ ID NO: 6. This sequence is absent from clones
MCV350 and MCV339.
[0096] In one aspect, the invention provides a method for
predicting the risk of developing an MCV-associated disease in a
patient comprising the step of detecting a VLP as defined above in
a tissue sample obtained from said patient.
[0097] In one aspect, the invention provides a method for
diagnosing an MCV-associated disease in a patient comprising the
step of detecting a VLP as defined above in a tissue sample
obtained from said patient.
[0098] Typically said tissue sample may be a blood sample, a urine
sample or a biopsy. In a preferred embodiment said tissue sample is
a biopsy, preferably a skin biopsy.
[0099] As used herein, the expression "MCV-associated disease"
refers to a disease in which MCV is a causative agent. An
MCV-associated disease can be primary MCV infection or a cancer
(such as Merkel cell carcinoma, small cell lung carcinoma, or other
carcinoma associated with MCV infection)
[0100] In a preferred embodiment said cancer is Merkel Cell
Carcinoma (MCC).
[0101] In another embodiment, said cancer is not Merkel Cell
Carcinoma (MCC).
[0102] The invention also relates to a kit for diagnosis an
MCV-associated disease in a patient comprising an anti-MCV agent as
defined above.
[0103] Typically, said kit can comprise an antibody which
specifically recognizes an antigen comprised between amino acids
456 to 817 of SEQ ID NO: 6 and means for revealing said
antibody.
Screening Method of the Invention
[0104] The invention also relates to a method for identifying an
agent that attenuates MCV infection.
[0105] In another embodiment, the invention provides a method of
identifying an agent that attenuates MCV infection, comprising the
step of exposing a target DNA to a polypeptide as defined above in
the presence or absence of a test compound.
[0106] In this context, attenuation can involve the reduction of
likelihood of infection, or reduction in magnitude. In some
applications, the reduction can amount to complete prophylaxis. In
accordance with this method, target DNA is exposed to a MCV
polypeptide (e.g., VP1, VP2, VP3, LT or ST). The target DNA should
include a sequence to which the MCV polypeptide can specifically
bind relative a negative control DNA. The assay is conducted in the
presence of a test agent, which is a putative agent under
investigation to assess whether it can attenuate the MCV infection.
Thus, the MCV polypeptide and the target DNA are exposed to each
other under conditions which, except for the test substance, are
suitable for the MCV polypeptide and target DNA to bind. It will be
understood that, as a result of this assay, the ability of the test
substance to attenuate binding of the MCV protein to the target DNA
identifies the test substance as a candidate agent for use as an
anti-MCV therapeutic agent. An example of this type of assay is a
gel-shift assay, which is known to those of ordinary skill in the
art. Also, while the test agent can be identified as a candidate
MCV therapeutic agent by this method, other tests likely will be
needed to assess whether the agent is safe and effective for
clinical use.
Methods of Treatment and Pharmaceutical Compositions of the
Invention
[0107] In some aspects the invention involves prophylactic and
therapeutic methods against an MCV-associated disease.
[0108] The invention also relates to a pharmaceutical composition
comprising a virus, a nucleic acid and/or a protein of the
invention.
[0109] The invention therefore relates to a pharmaceutical
composition comprising one or several of the elements as defined
above and a pharmaceutically acceptable carrier.
[0110] In particular, the invention relates to a pharmaceutical
composition comprising: [0111] a VLP according to the invention
and/or [0112] a nucleic acid according to the invention and/or
[0113] a polypeptide according to the invention and/or [0114] an
anti-MCV agent according to the invention and/or [0115] an agent
that attenuates MCV infection obtainable by the method as defined
above and a pharmaceutically acceptable carrier.
[0116] Suitable pharmaceutical compositions can be formulated for
delivery by oral, nasal, transdermal, parenteral, or other routes
by standard methodology. In this respect, the excipient can include
any suitable excipient (e.g., lubricant, diluent, buffer,
surfactant, co-solvent, glidant, etc.) known to those of ordinary
skill in the art of pharmaceutical compounding (see, e.g.,
"Handbook of Pharmaceutical Excipients" (Pharmaceutical Press),
Rowe et al, 5th Ed. (2006)).
[0117] In another aspect, the invention relates to prophylactic and
therapeutic methods against MCV-associated diseases. In this
context, the MCV-associated disease can be primary MCV infection or
a carcinoma (such as Merkel cell carcinoma, small cell lung
carcinoma, or other carcinoma associated with MCV infection). For
example, the invention provides a method of vaccinating a patient
against an MCV-associated disease. In accordance with this method,
a patient is vaccinated with MCC DNA and/or a MCV polypeptide under
conditions suitable for the patient to generate an immune response
to the MCV DNA and/or MCC polypeptide. A preferred agent for
serving as the vaccine is a polypeptide comprising at least 10, and
preferably at least the majority of, contiguous amino acids from
amino acids 456 to 817 of SEQ ID NO: 6. Another preferred agent is
a VLP as herein described. Indeed, rabbits and mice immunized with
an MCV VLP can exhibit very high anti-MCV antibody responses, with
50% neutralizing titers in the million-fold dilution range. It will
be understood that VLP could be combined with other viral subunit
vaccines such as the current vaccines against hepatitis B virus and
human papillomavirus, for combined vaccination protocols.
[0118] In another aspect, the invention provides a method for
treating a patient suffering from an MCV-associated disease
involving adoptive immunotherapy. In accordance with this method, a
population of T lymphocytes is first obtained from the patient.
Thereafter, the population of T lymphocytes is exposed ex vivo to
an MCV polypeptide, including a virus (such as described herein)
under conditions suitable to activate and expand the population of
T lymphocytes. For example, the T lymphocytes can be exposed to
cells in vitro, which express an MCV protein (e.g., having been
transfected with an expression cassette encoding the MCV
polypeptide). A preferred MCV protein includes at least 10, and
preferably at least the majority of, contiguous amino acids from
amino acids 456 to 817 of SEQ ID NO: 6. In other aspects, the
method can be practices using standard techniques (see, e.g., June,
J. Clin. Invest., 117(6) 1466-76 (2007)). After they have been
activated, at least some of the T lymphocytes are re-introduced
into the patient. Such a method can attenuate the severity of the
MCV-associated disease within the patient. It should be understood
that the method need not eradicate the MCV-associated disease
within the patient to be effective as a therapy. The method can be
deemed effective if it lessens symptoms, improves prognosis, or
augments other modes of therapy if used adjunctively.
[0119] It is believed that the newly-discovered MCV should respond
to agents that interfere with the replication of other
polyomaviruses. Thus, the invention provides a method of treating
an MCV-associated disease by administering such an agent to a
patient suffering from an MCV-associated disease. As noted, the
MCV-associated disease can be primary MCV infection, Merkel cell
carcinoma, small cell lung carcinoma, or another carcinoma that is
caused by MCV. It is believed that the administration of some such
agents can attenuate the severity of the MCV-associated disease
within the patient. Examples of such agents are cidofovir and
vidarabine, and other agents that interfere with polyomavirus
replication known to those of ordinary skill may be useful in
treating such conditions as well. Additional agents include
interferons and mTOR inhibitors (e.g., sirolimus and
tacrolimus).
[0120] The invention will be further described by the following
examples, which are not intended to limit the scope of the
protection defined by the claims.
[0121] FIG. 1: Schema of the main functional domains of ST and LT
proteins.
[0122] The bottom line shows the corresponding nucleotide positions
in the MCV genome. Arrows indicate the position of the interruption
of integrated viral DNA sequences determined by DIPS-PCR at the 3'
end () or the 5' end () of the viral genome. .dwnarw. refers to
mutation leading to the stop codon identified by sequencing. The
numbers above the arrows correspond to case number.
[0123] CR1: Conserved Region 1; OBD: Origin Binding Domain
EXAMPLES
Methods
Cases and Tumour Specimens
[0124] Ten cases of MCC were accumulated from 1996 to 2007. For 9
cases, a tumour specimen was fixed in formalin for histological
analysis and another specimen frozen in liquid nitrogen then kept
at -70.degree. C. for molecular studies. In one case, a patient
with MCC from the nasal septum (N.sup.o 4), only a fine needle
aspiration product of a supra-clavicular lymph node was available
for cytological analysis and DNA extraction. Twelve tumour
specimens were analysed from these 10 patients, corresponding to
primary tumours in 6 cases, 3 skin metastases and 3 lymph node
metastases.
[0125] According to the French regulation, patients were informed
of researches performed using the biological specimens obtained
during their treatment and did not express opposition.
Histological Analysis
[0126] Tumours were analysed according to standard histological
procedure. Histological reports specified the architectural pattern
as solid/cohesive (massive or trabecular) or diffuse/discohesive
[3] Immunohistochemistry was performed to confirm the diagnosis
using antibodies directed against chromogranin A (clone DAK-A3,
dilution: 1/200; Dako, Glostrup, Denmark) and synaptophysin (clone
SY38, dilution 1/40; Dako), markers expressed by virtually all MCCs
[4, 17]. Reactivity was scored as follows: 1: <10% of reactive
cells; 2: 10-50%; 3: >50%. Detection of cytokeratin intermediate
filaments was performed using pan anti-cytokeratins (clone KL1,
dilution 1/200; Beckman Coulter, Villepinte, France). Staining was
revealed by the Avidin Biotin technique, using DAB as a chromogen
(Dako).
MCV350 DNA Sequences Screening and Viral Load
[0127] MCV350 sequences were detected by PCR amplification with
primers MCV_ST_A and MCV_ST_B specific for the ST sequences (size
product 165 bp), MCV_LT_C and MCV_LT_D for the LT sequences (162
bp), and MCV_VP1_A and MCV_VP1_B for VP1 gene (204 bp). The viral
load was obtained by amplification of DNA (10 ng) with 600 nM of
each MCV_LT_C and MCV_LT_D primers in the SYBR Green PCR master mix
(Applied Biosystems, Courtaboeuf, France), using the standard
cycling conditions of 10 min at 95.degree. C. and 40 cycles (15 s
at 95.degree. C., 1 min at 60.degree. C.). Amplification of a
genomic DNA sequence ZNF277 (7q31.1) with primers IC5A and IC5B was
used as DNA quality control and reference for two copies of DNA
sequences per cell. Viral copy number was estimated by quantitative
PCR using a delta-delta Ct method [18].
[0128] In non MCC tumours, a total of 1277 DNA specimens from
tumours of various histological types and organs were obtained from
the DNA bank of the Institut Curie. DNA quality control assessed by
amplification of the ZNF277 DNA sequences showed that DNA quality
was insufficient in 36 cases which were discarded from the study.
The 1241 remaining specimens were analysed for MCV sequences using
MCV_LT_C and MCV_LT_D primers designed in the 5' part of the LT
sequences. See supplementary file for primer sequences.
MCV Cloning and FISH Experiments
[0129] The whole viral genome could be amplified in one case
(n.degree. 5) using primers MCV-U2 and MCV-L2 located at bases 5283
and 5282 of the MCV genome, respectively. DNA (250 ng) was
amplified by PCR (final volume 25 .mu.l) using the Expand 20
kb.sup.plus PCR System (Roche Applied Science, Meylan, France). The
viral genome (5387 bp) was then cloned in the pCR.RTM.-XL-TOPO.RTM.
vector (Invitrogen, Carlsbad, Calif. 92008) and sequenced. One of
the clones isolated (MCV-IC13) proved to encompass the whole viral
genome without any mutation likely to interrupt the coding
sequences. This genome was used for fluorescent in situ
hybridization (FISH) experiments. DNA was labelled by Nick
translation using the BioNick.TM. Labelling System (Invitrogen)
with biotinylated dATP. Hybridization was performed on frozen
histological sections. The slides were analysed using a Leica DMRB
microscope fitted with Quips (Visys, Downers Grove, Ill. 60515)
Image Capture System.
Viral Integration Sites
[0130] The DIPS-PCR technique, which allows the amplification of
genomic viral-cellular junctions [19], was used to investigate the
integration sites of MCV in MCC. After tumour DNA digestion with
restriction enzyme Taq1, enzyme-specific adapters were ligated to
the restriction fragments. The ligation products obtained were
subjected to PCR amplification which consisted of a first round of
linear PCR with a viral specific primer a, followed by a second
round of exponential PCR with a viral specific primer b, internal
to the previous one, and a second primer AP1 specific for the
adapter. The 3' viral-cellular DNA junctions were detected with
primers f_a and f_b and the cellular-5' viral DNA junctions with
primers r_a and r_b. PCR products were excised from an agarose gel,
purified and sequenced (see supplementary file a for primer
sequences). Sequences were submitted to database (UCSC Genome
Browser website; Working Draft March 2006) for genomic
localisation.
Viral Gene Expression
[0131] Total RNA was isolated using Trizol reagent (Invitrogen).
DNAse digestion using the Nucleospin RNA/Protein kit
(Macherey-Nagel, Hoerdt, France) was performed. Total RNA (1 .mu.g)
was reverse transcribed (RT+) using the GeneAmp RNA PCR Core Kit
(Applied Biosystems). For each sample, a negative control without
reverse transcriptase (RT-) was performed to verify the absence of
contaminating DNA. PCRs were performed in parallel on the RT+ and
RT- products. One hundredth of the RT+ or RT- product was used for
each PCR reaction (final volume of 25 .mu.l), in the presence of
600 nM of each specific primer and in the SYBR Green PCR master mix
(Applied Biosystems). Primers MCV_ST_A and MCV_ST_B were used for
MCV ST expression, MCV_LT_C and MCV_LT_D for LT expression.
MCV_ST_B primer was designed in the spliced LT sequences and thus
allows the amplification of ST sequences only. A12 and A13 primers
were used for the TATA Box Binding Protein (TBP) gene as a
reference for gene expression level. PCR amplifications were
performed in an ABI PRISM 7500 (Applied Biosystem). MCV350 mRNA
expression levels were directly compared to TBP expression using
the delta-delta Ct method [18].
MYC and IL20RA Gene Expression.
[0132] PCR with primers A227 and A228 for MYC expression and
IL20RA_B and IL20RA_C for IL20RA were performed in the conditions
previously described for the MCV350 genes.
Array-CGH.
[0133] Tumour cellularity of the samples was verified to be
>60%. Tumour DNA was prepared using DPNII digestion (Ozyme, St
Quentin-en-Yvelines, France), and purification on QIAquick column
(Qiagen, Courtaboeuf, France). Reference and test DNAs were
labelled with Cy3 and Cy5 cyanine dyes respectively (PerkinElmer,
Courtaboeuf, France) using the BioPrime random priming labelling
kit (Invitrogen). Reference and test DNA were precipitated together
with human Cot-1 DNA (Invitrogen), resuspended in hybridization
buffer, and denatured. The DNA was hybridised onto a genome-wide
DNA microarray consisting of 5K BAC clones spotted in triplicate,
with a 1 Mbase resolution (CIT/INSERM U830/IntegraGen). Slides were
scanned using an Axon GenePix 4000B scanner (Molecular Devices,
Sunnyvale, Calif.). Image analysis was performed with the Axon
GenePix 5.cndot.1 software (Molecular Devices). The data was
visualized using the VAMP software [20].
Results
Patients and Tumours
[0134] Ten cases of Merkel cell carcinoma (MCC) in 8 male and 2
female patients with a mean age of 79.1 (63-85) were studied (table
2). All primary tumours were dermal, localised on the head and neck
in 4 cases, the limbs in 4 cases, and the trunk in 2 cases (table
2). In one case (N.sup.o 6), two cutaneous metastases were analysed
in addition to primary tumour. The mean primary tumour size was
23.9 mm (10 to 45 mm) Histological analysis showed the
architectural pattern to be solid in 5 cases, either trabecular (3
cases) or massive (2 cases) and discohesive/diffuse in 4 cases.
This latter pattern was characterised by a proliferation of tumour
cells that lack cohesion and appear individually dispersed
throughout the connective tissue (data not shown). All 9 cases
exhibited the co-expression of chromogranin A and synaptophysin
neuro-endocrine markers, a characteristic immunophenotype of MCC
(data not shown and table 2). Cytokeratins staining disclosed a
dot-like immunolabelling close to the nucleus of tumour cells,
corresponding to a localised aggregate of these intermediate
filaments (data not shown).
TABLE-US-00002 TABLE 2 Clinical and histological data in Merkel
cell carcinoma histology localisation size immunophenotype Case age
Sex primary metastasis (mm) pattern chromogranin synaptophysin 1 82
M eyelid cervical LN* 30 discohesive/diffuse 3 1 2 72 M forearm* 10
discohesive/diffuse 2 2 3 84 M thigh inguinal LN* 27
discohesive/diffuse 3 2 4 79 F nose cervical LN* 30
solid/trabecular ND ND 5 81 M ankle leg* 11 solid/trabecular 2 2 6a
80 M wrist* 21 solid/massive 3 3 6b trunk* -- ND ND ND 6c trunk* --
ND ND ND 7 82 F cheek* 15 solid/massive 1 1 8 85 M buttock* 30
discohesive/diffuse 3 2 9 63 M breast* 20 solid/trabecular 2 3 10
83 M ear* 45 solid/massive 3 3 LN: lymph node *specimens analysed
for MCV characterisation
TABLE-US-00003 TABLE 3 Viral and genetic data in Merkel cell
carcinoma Putative target genes MCV RNA Array-CGH expression MCV
Viral expression level chromosome Chromosome MCV Putative level
Case DNA load* small T large T imbalances** insertion sites
Locus*** breakpoints target genes MYC IL20RA 1 + 3 0.23 3.58 +5p,
-5q, -8p, 8q24.21 130177462 1532 (3') MYC 0.008 0.000 2 + 1.2 0.42
3.25 no imbalance 12q23.1 97372542 5202 (3') AX747640 0.007 0.000 3
+ 0.6 ND ND +6p, +11, +17p 2q32.3 196674834 3925 (5') -- -- 4 + 3.3
ND ND -2, +6, -7, 20q11.21 31474040 3712 (3') SNTA1 -- -- -10, -17
5 + 62.2 0.24 2.62 +1p, +1q 4q13.1 64683073 1515 (3') SRD5A2L2
0.043 0.073 6a + 3.8 3.16 21.56 no imbalance 3q26.33 183625703 2663
(3') ATP11B 0.025 0.000 6b + ND ND ND ND 3q26.33 183625703 2663
(3') -- -- -- 6c + ND ND ND ND 3q26.33 183625703 2663 (3') -- -- --
7 + 1.7 0.21 1.46 +1q 5q35.1 170684993 1978 (5') TLX3 0.034 0.000 8
+ 6.3 0.28 3.86 +11 ? 3119 (5') 0.012 0.000 9 + 1 0.33 3.36 no
imbalance 6q23.3 137409329 2240 (3') IL20RA 0.036 0.029 137408299
3305 (5') 10 + 10.3 0.39 5.50 +1q, +6p, Yq12 57288464 2980 (3')
0.068 0.000 -6qter, +7pter *number of equivalent viral genome per
cell; **recurrent imbalances in bold; ***Working Draft march
2006
TABLE-US-00004 TABLE 4 MCV analysis in non MCC human tumours Nb of
MCV Organs Histological tumour type cases DNA Skin Basal cell
carcinoma 13 -- Melanoma 13 -- Others 2 -- Normal skin 4 -- Uterine
cervix Invasive carcinoma HPV positive 26 -- HPV negative 18 --
Large bowel Adenocarcinoma 38 -- Others 1 -- Liver Metastatic
melanoma 94 -- Metastatic breast carcinoma 16 -- Others 4 -- Uveal
tract Melanoma 45 -- Ovary Serous adenocarcinoma 71 -- Endometrioid
carcinoma 9 -- Mucinous carcinoma 3 -- Clear cell carcinoma 5 --
Poorly differentiated carcinoma 32 -- Metastatic carcinoma 39 --
Serous border line tumours 6 -- Others 7 -- Breast Invasive ductal
carcinoma 451 -- Invasive lobular carcinoma 49 -- Poorly
differentiated carcinoma 41 -- Medullary carcinoma 9 -- Mucinous or
papillary carcinoma 18 -- Axillary node metastases 31 --
Intraductal carcinoma 41 -- Phyllodes tumor 41 -- Others 6 -- Bone
& soft tissue Ewing tumor 30 -- Rhabdomyosarcoma 25 --
Desmoplastic tumor 24 -- Neuroblastoma 21 -- Fibromatosis 4 --
Others 4 -- 1241
MCV350 DNA Sequences in Merkel Cell Carcinoma.
[0135] DNA extracted from frozen tumour tissue or cells was
analysed by PCR for the presence of MCV350 DNA sequences using
primers designed in the ST, LT, or VP1 sequences (cf supplementary
data). All 10 cases of MCC were positive for MCV (table 3). DNA
fragments with the expected sizes of 165, 162 and 204 bp were
obtained in each case except in case N.sup.o 1 for which only DNA
corresponding to the ST and LT sequences could be amplified.
[0136] Q-PCR experiments using amplimers designed in the LT
sequences of the MCV350 were performed to assess the number of
viral genomes per cell in MCC. Viral DNA loads ranging from 0.6 to
10.3 genome-equivalent per carcinoma cell were observed in 9 cases.
A much higher viral load of 62.2 was detected in one case (N.sup.o
5) (table 3) which was further analysed using contiguous and
inversely oriented primers. This experiment allowed the
amplification of the whole viral genome (5387 bp), suggesting the
presence of viral episomes. This genome was cloned. Sequencing
showed that, in one of the clones isolated (MCV-IC13), ST, LT and
VPs viral genes were fully conserved without mutation that could
lead to truncated protein. This sequence showed a 99.3% identity
rate with that of MCV350.
[0137] In order to verify that MCV 350 DNA sequences were located
in the nucleus of epithelial tumour cells, we performed in situ
hybridization analysis using the whole viral genome as a probe.
FISH experiments were performed on frozen sections of case 8 which
contains 6 copies of the MCV genome integrated at a single site. A
single fluorescent signal was observed in the nucleus of epithelial
tumour cells (data not shown). About 90% of the cells showed the
signal, corresponding to the clonal pattern of integration. No
significant signal was found in non tumour cells.
MCV350 DNA Sequences in Non Merkel Cell Tumours
[0138] To determine whether MCV DNA sequences were present in
tumour types other than MCC, detection of MCV350 sequences was
performed by PCR using primers designed in the 5' part of the LT
sequences found to be conserved in all MCC cases. A total of 1241
specimens, taken from tumours of epithelial or mesenchymal origin,
developed in adults or children, were included in the analysis
(table 4). In none of these 1241 different specimens was there
evidence of DNA likely to correspond to MCV sequences.
MCV Genes Expression in Merkel Cell Carcinoma
[0139] Frozen tissue specimens from 8 of our 10 cases of MCC were
available for RNA extraction. Total RNA was treated with DNAse to
avoid the amplification of viral DNA. Quantitative RT-PCR was
performed using amplimers designed in ST and LT sequences.
Expression level of the TBP human gene was used as reference and
three cervical cancer cell lines (IC1, 2, 3) negative for MCV were
used as control. RNAs from the MCV sequences were expressed in all
8 cases. LT expression level ranged from 1.46 to 21.56 fold TBP and
ST from 0.21 to 3.16 (table 3). No significant correlation between
viral DNA load and viral RNA expression level was observed.
Chromosome Localization of the Viral DNA Sequences.
[0140] Integration of viral DNA sequences into the tumour cell
genome was investigated using the DIPS-PCR method which allows the
localisation of the viral integration site at the molecular level.
The integration site was identified in all 10 cases with primers
designed to determine either the 3' or the 5' virus-host junctions.
All cases harboured a single integration site which was found in 10
different loci (table 3). Viral DNA sequences were found inserted
in the long arm of chromosomes 2, 3, 4, 5, 6, 8, 12, 20 and Y. In
case N.sup.o 6, the same chromosome localisation was observed in
the primary and in the two skin metastases, demonstrating the
clonality of the insertional mutation. In case N.sup.o 8, the viral
genome was interrupted at base 3119, at the junction between LT and
VP1 sequences. Only 70 bp of cell DNA at the 5' virus-host junction
could be amplified. When compared to the human database, the
specificity of this short sequence was not sufficient to correspond
to a unique locus.
[0141] Analysis of the virus-host junctions allowed specification
of the pattern of the integrated viral sequences. In 6 cases
(N.sup.o 1, 5, 6, 7, 9, 10), the virus-host junction was located in
the 3' part of the LT sequences (between nt 1515 and nt 2980) which
were thus partly deleted (FIG. 1). In two cases (N.sup.o 4, 2), the
3' virus-host junction was located in VP1 (nt 3712) or in the
regulatory region (nt 5202) and the LT sequences were fully
conserved. LT DNA sequencing showed the presence of a 72 bp
deletion (1403-1480) leading to a stop codon (PY261X) (case N.sup.o
2) and the presence of a mutation (1390) leading to a stop codon
(PQ255X) (case N.sup.o 4). In two cases (N.sup.o 3, 8), only the 5'
host-virus junction was identified and the localisation of the 3'
break point regarding LT sequences could not be specified. In all 8
informative cases, the 3' part of integrated viral LT sequences was
prematurely truncated (FIG. 1). In all 10 cases, ST sequences were
fully conserved.
Status of Cellular Genes Potentially Involved in Oncogenesis and
Located at the Vicinity of the Integration Sites.
[0142] The possibility that integration of MCV DNA could lead to
the deregulation of cellular genes involved in the tumour process
was investigated. The genes located in the vicinity of the
integrated viral sequences were identified. Viral sequences were
found to be located in the AX747640 and SNAT1 genes (cases N.sup.o
2 and N.sup.o 3), at 1.3 kb from the IL20RA gene (case N.sup.o 9),
at 1 Mb from the SRD5A2L2 gene (case N.sup.o 5) and at 1.35 Mb from
MYC (case N.sup.o 1) (table 3). Since MYC has been found activated
by viral insertion in human tumours [21] and IL20 RA inactivation
implicated in lung carcinogenesis [22] the expression level of
these genes was further assessed by RT-PCR for 8 of the 10 cases.
No significant gene deregulation related to MCV viral insertion was
found (table 3).
Array-CGH Analysis
[0143] Cellular DNA sequence copy number changes are reported in
table 3. Three of the 10 samples analysed did not show any
imbalance. Recurrent imbalances were gains of 1q (2 cases), 6p (3
cases), and 1l (2 cases), and loss of 17p (2 cases). No correlation
was found between these chromosome rearrangements and integration
sites of the MCV.
REFERENCES
[0144] Throughout this application, various references describe the
state of the art to which this invention pertains. The disclosures
of these references are hereby incorporated by reference into the
present disclosure. [0145] 1. Toker C. Trabecular carcinoma of the
skin. Arch Dermatol 1972; 105:107-110 [0146] 2. Sibley R K, Rosai
J, Foucar E, Dehner L P and Bosl G. Neuroendocrine (Merkel cell)
carcinoma of the skin. A histologic and ultrastructural study of
two cases. Am J Surg Pathol 1980; 4:211-221 [0147] 3. Frigerio B,
Capella C, Eusebi V, Tenti P and Azzopardi J G. Merkel cell
carcinoma of the skin: the structure and origin of normal Merkel
cells. Histopathology 1983; 7:229-249 [0148] 4. Llombart B,
Monteagudo C, Lopez-Guerrero J A, Carda C, Jorda E, Sanmartin O, et
al. Clinicopathological and immunohistochemical analysis of 20
cases of Merkel cell carcinoma in search of prognostic markers.
Histopathology 2005; 46:622-634 [0149] 5. Swann M H and Yoon J.
Merkel cell carcinoma. Semin Oncol 2007; 34:51-56 [0150] 6. Hodgson
N C. Merkel cell carcinoma: changing incidence trends. J Surg Oncol
2005; 89:1-4 [0151] 7. Lemos B and Nghiem P. Merkel cell carcinoma:
more deaths but still no pathway to blame. J Invest Dermatol 2007;
127:2100-2103 [0152] 8. Feng H, Shuda M, Chang Y and Moore P S.
Clonal integration of a polyomavirus in human Merkel cell
carcinoma. Science 2008; 319:1096-1100 [0153] 9. Kassem A,
Schopflin A, Diaz C, Weyers W, Stickeler E, Werner M, et al.
Frequent detection of Merkel cell polyomavirus in human Merkel cell
carcinomas and identification of a unique deletion in the VP1 gene.
Cancer Res 2008; 68:5009-5013 [0154] 10. Becker J C, Houben R,
Ugurel S, Trefzer U, Pfohler C and Schrama D. MC polyomavirus is
frequently present in Merkel cell carcinoma of European patients. J
Invest Dermatol 2009; 129:248-250 [0155] 11. Garneski K M, Warcola
A H, Feng Q, Kiviat N B, Leonard J H and Nghiem P. Merkel cell
polyomavirus is more frequently present in North American than
Australian Merkel cell carcinoma tumors. J Invest Dermatol 2009;
129:246-248 [0156] 12. Gardner S D, Field A M, Coleman D V and
Hulme B. New human papovavirus (B.K.) isolated from urine after
renal transplantation. Lancet 1971; 1:1253-1257 [0157] 13. Padgett
B L, Walker D L, ZuRhein G M, Eckroade R J and Dessel B H.
Cultivation of papova-like virus from human brain with progressive
multifocal leucoencephalopathy. Lancet 1971; 1:1257-1260 [0158] 14.
Allander T, Andreasson K, Gupta S, Bjerkner A, Bogdanovic G,
Persson M A, et al. Identification of a third human polyomavirus. J
Virol 2007; 81:4130-4136 [0159] 15. Gaynor A M, Nissen M D, Whiley
D M, Mackay I M, Lambert S B, Wu G, et al. Identification of a
novel polyomavirus from patients with acute respiratory tract
infections. PLoS Pathog 2007; 3:e64 [0160] 16. zur Hausen H. Novel
human polyomaviruses--re-emergence of a well known virus family as
possible human carcinogens. Int J Cancer 2008; 123:247-250 [0161]
17. Asioli S, Righi A, Volante M, Eusebi V and Bussolati G. p63
expression as a new prognostic marker in Merkel cell carcinoma.
Cancer 2007; 110:640-647 [0162] 18. Livak K J and Schmittgen T D.
Analysis of relative gene expression data using real-time
quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;
25:402-408 [0163] 19. Luft F, Klaes R, Nees M, Durst M, Heilmann V,
Melsheimer P, et al. Detection of integrated papillomavirus
sequences by ligation-mediated PCR (DIPS-PCR) and molecular
characterization in cervical cancer cells. Int J Cancer 2001;
92:9-17 [0164] 20. La Rosa P, Viara E, Hupe P, Pierron G, Liva S,
Neuvial P, et al. VAMP: visualization and analysis of array-CGH,
transcriptome and other molecular profiles. Bioinformatics 2006;
22:2066-2073 [0165] 21. Peter M, Rosty C, Couturier J, Radvanyi F,
Teshima H and Sastre-Garau X. MYC activation associated with the
integration of HPV DNA at the MYC locus in genital tumors. Oncogene
2006; 25:5985-5993 [0166] 22. Tessema M, Willink R, Do K, Yu Y Y,
Yu W, Machida E O, et al. Promoter methylation of genes in and
around the candidate lung cancer susceptibility locus 6q23-25.
Cancer Res 2008; 68:1707-1714.
Sequence CWU 1
1
1215387DNAMerkel cell Polyomavirus MCV-IC13 1ggggctccta gcctccgagg
cctctggaaa aaaaagagag aggcctctga ggcttaagag 60gcttaattag caaaaaaggc
agtatctaag ggcagatccc aagggcggga aactgcagta 120taaaaaccac
tccttagtga ggtagctcat ttgctcctct gctctttctg caaactcctt
180ctgcatatag acaagatgga tttagtccta aataggaaag aaagagaggc
tctctgcaag 240cttttagaga ttgctcctag ttgttatggc aacatccctc
tgatgaaagc tgctttcaaa 300agaagctgct taaagcatca ccctgataaa
gggggaaatc ctgttataat gatggaattg 360aacacccttt ggagcaaatt
ccagcaaaat atccacaagc tcagaagtga cttctctatg 420tttgatgagg
tcagtacaaa atttccttgg gaagaatatg gaactttaaa ggattatatg
480caaagtggat ataatgctag attttgcaga ggtcctgggt gcatgcttaa
gcaacttaga 540gattctaagt gcgcttgtat tagctgtaag ttgtctcgcc
agcattgtag tctaaaaact 600ttaaagcaaa aaaactgtct gacgtgggga
gagtgttttt gctatcagtg ctttattctt 660tggtttggat ttcctcctac
ttgggaaagt tttgactggt ggcaaaaaac tttagaagaa 720actgactact
gcttactgca tctgcacctt ttctagactc ctacttcctt cctctgtaag
780tattagatat ggaaaagtct ataaggcaaa atatcaaaga aaggttattt
atgacagatt 840ttctgtactt tcccatctag gttgacgagg cccctatata
tgggaccact aaattcaaag 900aatggtggag atcaggagga ttcagcttcg
ggaaggcata cgaatatggg cccaatccac 960acgggaccaa ctcaagatcc
agaaagcctt cctccaatgc atccagggga gcccccagtg 1020gaagctcacc
accccacagc cagagctctt cctctgggta tgggtccttc tcagcgtccc
1080aggcttcaga ctcccagtcc agaggacccg atatacctcc cgaacaccat
gaggaaccca 1140cctcatcctc tggatccagt agcagagggg agaccaccaa
ttcaggaaga gaatccagca 1200cacccaatgg aaccagtgta cctagaaatt
cttccagaac ggatggcacc tgggaggatc 1260tcttctgcga tgaatcactt
tcctcccctg agcctccctc gtcctctgag gagcctgagg 1320agcccccctc
ctcaagaagc tcgccccggc agcccccgtc ttcctctgcc gaggaggcct
1380catcatctca gtttacagat gaggaataca gatcctcctc cttcaccacc
ccgaagaccc 1440ctcctccatt ctcaagaaag cgaaaatttg gggggtcccg
aagctctgca agctctgcta 1500gttcagcaag ttttacaagc actccaccaa
agccaaaaaa gaacagagaa actcctgttc 1560ctactgattt tcctattgat
ctttctgatt atcttagcca tgctgtatat agtaataaaa 1620cagtaagttg
ttttgccatt tatactactt ctgataaagc tatagagtta tatgataaga
1680ttgagaaatt taaagttgat tttaaaagca ggcatgcctg tgaattagga
tgtattttat 1740tgtttataac tttatcaaag catagagtat ctgctattaa
gaatttttgc tctaccttct 1800gcactataag ctttttaatt tgtaaaggag
tgaataagat gcctgaaatg tataataatt 1860tatgcaagcc cccttacaaa
ttactgcaag agaataagcc actgctcaat tatgaatttc 1920aagaaaaaga
aaaagaggcc agctgtaatt ggaatttagt tgctgaattt gcttgtgaat
1980atgagctaga cgaccacttt attatcttag cccattatct agactttgca
aaaccatttc 2040cttgccaaaa gtgtgaaaat agatctcgcc tcaaacctca
caaggctcat gaggctcatc 2100attctaatgc taagctattt tatgaatcta
aatctcagaa aaccatttgc caacaagccg 2160cagacactgt tctagccaaa
aggaggttag agatgctgga aatgaccagg acagaaatgc 2220tatgtaagaa
gtttaagaag cacctagaga gattaagaga tttagataca atagatctac
2280tgtattatat gggtggtgtg gcctggtact gctgcttatt tgaagagttt
gaaaagaagc 2340tgcagaaaat tattcaatta ttaacagaga atatacctaa
gtatagaaac atttggttta 2400aagggcctat taacagtgga aaaacaagct
ttgctgcagc cttaatagat ttgctagaag 2460ggaaggcctt gaatataaac
tgtccatctg ataaactgcc ttttgaacta ggatgtgctt 2520tggataaatt
tatggttgtt tttgaggatg tgaaagggca aaatagccta aataaagatc
2580tgcaaccagg gcaaggaata aataaccttg ataacttaag agatcatcta
gatggtgctg 2640tagctgtaag cttagagaag aagcatgtga ataaaaagca
tcagattttt cctccttgta 2700ttgttactgc taatgattat tttattccca
aaacattaat agcaagattt agttatactt 2760tacacttttc cccaaaggca
aatctaagag attccctgga tcagaacatg gaaataagaa 2820aaagaagaat
tcttcaaagt ggaaccactt tattgctttg tcttatttgg tgcttgcctg
2880atacaacctt taagccttgc ttacaagaag aaattaaaaa ctggaagcaa
attttacaga 2940gtgaaatatc atatggtaaa ttttgtcaaa tgatagaaaa
tgtagaagct ggtcaggacc 3000ctctgctcaa tattcttatt gaggaagagg
gccctgagga aactgaagaa acccaagatt 3060ctggtacttt ttctcaataa
aggcatctgc ttcatatttc ctgtgtttgt ttttctgggg 3120cctacttaac
tgaataggaa tgcatgaaat aattctcata attcttgtgt ttggctttct
3180ttttgagagg ccttttgagg tcctttcagt ggcgccttgc ccttatcctg
ctgattactt 3240tggaatgtta ctgctgctgg ggcaacagag ggctttgggt
aaacagtttt ctcctgccca 3300aatttatcta aaaatctgac aatatcagga
tcaccaggta attgttctga cccctcatat 3360attctaacct cttctacctg
attatctttt ccttccatag gttggcctga cacttttggc 3420attaagttgc
tgaagagtga gtttattaaa ttaactactg ggtaggggtt tttcacccat
3480ctttttctga aagtaacatt aaaatatcta ggcaacccat gaagagccat
ttttccactg 3540gttttaaaca gaaaccccac tatgtctgca cagctaataa
ataggccatc tcctttgcat 3600agagggccca ctccattctc atctaaaagg
acagtagtta gagtattact aaattgaaga 3660actgtaggag tctgagagcc
tgtctgaata gacccatagt atctactgtt ttcattttta 3720gaaggatcag
gacaccatac ttctatagga taatttccat ctttatctaa ttttgcttta
3780gcttgtggat ctaggccctg atttttaggt gtcatttttc ttcccaatac
agtttcaatt 3840gtaataggcc caccatttgt agtttttgga tactcagtct
ggtaatctaa aactaggcct 3900tgcaaatcca gaggttctcc cccaatggca
aacatatggt aatttactcc tgacacagga 3960ataccagcac cataatcatg
aactcttttc atgtcccaat aatgaacatt aattaaagaa 4020cttattccaa
ctacttctgt tttaacagat attgcctccc acatctgcaa tgtgtcacag
4080gtaatatcct catttagcat tggcagagac actcttgcca cactgtaagc
tggcaaattt 4140tccttgatgg gctgatctgg agatgatccc tttggctgca
ggtcataagt ataagtatac 4200cagtttgaag tagtaggaag atcaggggaa
ttaactccca ttcttggatt caaatacaac 4260tcaatttggg taatgctatc
ttctccagta accacagtta atacttccac tcctccttta 4320acaagcagtt
ttggaactga ggcaacatta gggcagcatc ccggcttagg tatacattgc
4380cttttgggtg ttttacaggt ggatgatgct tttctttttg gtgccatctt
caattacttg 4440taattcagga gaaatatatc cactaaggcc tagtaccaga
ggaagaagcc aatctggagt 4500ttgctgctgc agagttcctc ctatatgttc
aggaattaat atagcctctc ctgataaaag 4560gccctgattc tgagaagcag
ttgtgtgaaa gacccaccgg ctatttagta tcagattcac 4620taggtttgat
tgtatctgca gcctagaggt aggagataaa gaattaaaaa tattttgccc
4680cacagaatgc agcaagctat tttcccactg cagaggatct aggctaaagg
ccataagtgc 4740atgcctcaaa acctcattac tacctaccca cgaaacatcc
ctctttacaa gtgacacttg 4800ctcgcgtgac aacctcaccc ccacagttat
tagagagcct ataccactaa cagtttggag 4860aatgaagcca taagttaaac
cttggttaac caaagaagcc actaatgaga aatttgaaaa 4920ctgttcagct
gtgaacccaa gttgagctaa agcctcaatg ccagaaatac cctcaattgt
4980cattaaactg gagatctctg cttccaaagc tgctaaagct tctcctgtaa
gaatagcttc 5040caaagttact cctgtggtgg cacttagttc agtagcaatt
tcaccaatat tggccagcag 5100tgtgatgatg ccccccatcc tgaaaaataa
ataaggatac ttactctttt aatgtcctcc 5160tccctttgta agagaaaaaa
aagcctccgg gcctcccttg ttgaaaaaaa gttaagagtc 5220ttccgtctcc
ctcccaaaca gaaagaaaaa aagttttgtt tatcagtcaa actccgcctc
5280tccaggaaat gagtcaatgc cagaaaccct gcagcaataa aagttcaatc
atgtaaccac 5340aacttggctg cctaggtgac tttttttttt caagttggca gaggctt
53872561DNAMerkel cell Polyomavirus MCV-IC13 2atggatttag tcctaaatag
gaaagaaaga gaggctctct gcaagctttt agagattgct 60cctagttgtt atggcaacat
ccctctgatg aaagctgctt tcaaaagaag ctgcttaaag 120catcaccctg
ataaaggggg aaatcctgtt ataatgatgg aattgaacac cctttggagc
180aaattccagc aaaatatcca caagctcaga agtgacttct ctatgtttga
tgaggtcagt 240acaaaatttc cttgggaaga atatggaact ttaaaggatt
atatgcaaag tggatataat 300gctagatttt gcagaggtcc tgggtgcatg
cttaagcaac ttagagattc taagtgcgct 360tgtattagct gtaagttgtc
tcgccagcat tgtagtctaa aaactttaaa gcaaaaaaac 420tgtctgacgt
ggggagagtg tttttgctat cagtgcttta ttctttggtt tggatttcct
480cctacttggg aaagttttga ctggtggcaa aaaactttag aagaaactga
ctactgctta 540ctgcatctgc accttttcta g 5613186PRTMerkel cell
Polyomavirus MCV-IC13 3Met Asp Leu Val Leu Asn Arg Lys Glu Arg Glu
Ala Leu Cys Lys Leu1 5 10 15Leu Glu Ile Ala Pro Ser Cys Tyr Gly Asn
Ile Pro Leu Met Lys Ala 20 25 30Ala Phe Lys Arg Ser Cys Leu Lys His
His Pro Asp Lys Gly Gly Asn 35 40 45Pro Val Ile Met Met Glu Leu Asn
Thr Leu Trp Ser Lys Phe Gln Gln 50 55 60Asn Ile His Lys Leu Arg Ser
Asp Phe Ser Met Phe Asp Glu Val Ser65 70 75 80Thr Lys Phe Pro Trp
Glu Glu Tyr Gly Thr Leu Lys Asp Tyr Met Gln 85 90 95Ser Gly Tyr Asn
Ala Arg Phe Cys Arg Gly Pro Gly Cys Met Leu Lys 100 105 110Gln Leu
Arg Asp Ser Lys Cys Ala Cys Ile Ser Cys Lys Leu Ser Arg 115 120
125Gln His Cys Ser Leu Lys Thr Leu Lys Gln Lys Asn Cys Leu Thr Trp
130 135 140Gly Glu Cys Phe Cys Tyr Gln Cys Phe Ile Leu Trp Phe Gly
Phe Pro145 150 155 160Pro Thr Trp Glu Ser Phe Asp Trp Trp Gln Lys
Thr Leu Glu Glu Thr 165 170 175Asp Tyr Cys Leu Leu His Leu His Leu
Phe 180 1854234DNAMerkel cell Polyomavirus MCV-IC13 4atggatttag
tcctaaatag gaaagaaaga gaggctctct gcaagctttt agagattgct 60cctagttgtt
atggcaacat ccctctgatg aaagctgctt tcaaaagaag ctgcttaaag
120catcaccctg ataaaggggg aaatcctgtt ataatgatgg aattgaacac
cctttggagc 180aaattccagc aaaatatcca caagctcaga agtgacttct
ctatgtttga tgag 23452220DNAMerkel cell Polyomavirus MCV-IC13
5gttgacgagg cccctatata tgggaccact aaattcaaag aatggtggag atcaggagga
60ttcagcttcg ggaaggcata cgaatatggg cccaatccac acgggaccaa ctcaagatcc
120agaaagcctt cctccaatgc atccagggga gcccccagtg gaagctcacc
accccacagc 180cagagctctt cctctgggta tgggtccttc tcagcgtccc
aggcttcaga ctcccagtcc 240agaggacccg atatacctcc cgaacaccat
gaggaaccca cctcatcctc tggatccagt 300agcagagggg agaccaccaa
ttcaggaaga gaatccagca cacccaatgg aaccagtgta 360cctagaaatt
cttccagaac ggatggcacc tgggaggatc tcttctgcga tgaatcactt
420tcctcccctg agcctccctc gtcctctgag gagcctgagg agcccccctc
ctcaagaagc 480tcgccccggc agcccccgtc ttcctctgcc gaggaggcct
catcatctca gtttacagat 540gaggaataca gatcctcctc cttcaccacc
ccgaagaccc ctcctccatt ctcaagaaag 600cgaaaatttg gggggtcccg
aagctctgca agctctgcta gttcagcaag ttttacaagc 660actccaccaa
agccaaaaaa gaacagagaa actcctgttc ctactgattt tcctattgat
720ctttctgatt atcttagcca tgctgtatat agtaataaaa cagtaagttg
ttttgccatt 780tatactactt ctgataaagc tatagagtta tatgataaga
ttgagaaatt taaagttgat 840tttaaaagca ggcatgcctg tgaattagga
tgtattttat tgtttataac tttatcaaag 900catagagtat ctgctattaa
gaatttttgc tctaccttct gcactataag ctttttaatt 960tgtaaaggag
tgaataagat gcctgaaatg tataataatt tatgcaagcc cccttacaaa
1020ttactgcaag agaataagcc actgctcaat tatgaatttc aagaaaaaga
aaaagaggcc 1080agctgtaatt ggaatttagt tgctgaattt gcttgtgaat
atgagctaga cgaccacttt 1140attatcttag cccattatct agactttgca
aaaccatttc cttgccaaaa gtgtgaaaat 1200agatctcgcc tcaaacctca
caaggctcat gaggctcatc attctaatgc taagctattt 1260tatgaatcta
aatctcagaa aaccatttgc caacaagccg cagacactgt tctagccaaa
1320aggaggttag agatgctgga aatgaccagg acagaaatgc tatgtaagaa
gtttaagaag 1380cacctagaga gattaagaga tttagataca atagatctac
tgtattatat gggtggtgtg 1440gcctggtact gctgcttatt tgaagagttt
gaaaagaagc tgcagaaaat tattcaatta 1500ttaacagaga atatacctaa
gtatagaaac atttggttta aagggcctat taacagtgga 1560aaaacaagct
ttgctgcagc cttaatagat ttgctagaag ggaaggcctt gaatataaac
1620tgtccatctg ataaactgcc ttttgaacta ggatgtgctt tggataaatt
tatggttgtt 1680tttgaggatg tgaaagggca aaatagccta aataaagatc
tgcaaccagg gcaaggaata 1740aataaccttg ataacttaag agatcatcta
gatggtgctg tagctgtaag cttagagaag 1800aagcatgtga ataaaaagca
tcagattttt cctccttgta ttgttactgc taatgattat 1860tttattccca
aaacattaat agcaagattt agttatactt tacacttttc cccaaaggca
1920aatctaagag attccctgga tcagaacatg gaaataagaa aaagaagaat
tcttcaaagt 1980ggaaccactt tattgctttg tcttatttgg tgcttgcctg
atacaacctt taagccttgc 2040ttacaagaag aaattaaaaa ctggaagcaa
attttacaga gtgaaatatc atatggtaaa 2100ttttgtcaaa tgatagaaaa
tgtagaagct ggtcaggacc ctctgctcaa tattcttatt 2160gaggaagagg
gccctgagga aactgaagaa acccaagatt ctggtacttt ttctcaataa
22206817PRTMerkel cell Polyomavirus MCV-IC13 6Met Asp Leu Val Leu
Asn Arg Lys Glu Arg Glu Ala Leu Cys Lys Leu1 5 10 15Leu Glu Ile Ala
Pro Ser Cys Tyr Gly Asn Ile Pro Leu Met Lys Ala 20 25 30Ala Phe Lys
Arg Ser Cys Leu Lys His His Pro Asp Lys Gly Gly Asn 35 40 45Pro Val
Ile Met Met Glu Leu Asn Thr Leu Trp Ser Lys Phe Gln Gln 50 55 60Asn
Ile His Lys Leu Arg Ser Asp Phe Ser Met Phe Asp Glu Val Asp65 70 75
80Glu Ala Pro Ile Tyr Gly Thr Thr Lys Phe Lys Glu Trp Trp Arg Ser
85 90 95Gly Gly Phe Ser Phe Gly Lys Ala Tyr Glu Tyr Gly Pro Asn Pro
His 100 105 110Gly Thr Asn Ser Arg Ser Arg Lys Pro Ser Ser Asn Ala
Ser Arg Gly 115 120 125Ala Pro Ser Gly Ser Ser Pro Pro His Ser Gln
Ser Ser Ser Ser Gly 130 135 140Tyr Gly Ser Phe Ser Ala Ser Gln Ala
Ser Asp Ser Gln Ser Arg Gly145 150 155 160Pro Asp Ile Pro Pro Glu
His His Glu Glu Pro Thr Ser Ser Ser Gly 165 170 175Ser Ser Ser Arg
Gly Glu Thr Thr Asn Ser Gly Arg Glu Ser Ser Thr 180 185 190Pro Asn
Gly Thr Ser Val Pro Arg Asn Ser Ser Arg Thr Asp Gly Thr 195 200
205Trp Glu Asp Leu Phe Cys Asp Glu Ser Leu Ser Ser Pro Glu Pro Pro
210 215 220Ser Ser Ser Glu Glu Pro Glu Glu Pro Pro Ser Ser Arg Ser
Ser Pro225 230 235 240Arg Gln Pro Pro Ser Ser Ser Ala Glu Glu Ala
Ser Ser Ser Gln Phe 245 250 255Thr Asp Glu Glu Tyr Arg Ser Ser Ser
Phe Thr Thr Pro Lys Thr Pro 260 265 270Pro Pro Phe Ser Arg Lys Arg
Lys Phe Gly Gly Ser Arg Ser Ser Ala 275 280 285Ser Ser Ala Ser Ser
Ala Ser Phe Thr Ser Thr Pro Pro Lys Pro Lys 290 295 300Lys Asn Arg
Glu Thr Pro Val Pro Thr Asp Phe Pro Ile Asp Leu Ser305 310 315
320Asp Tyr Leu Ser His Ala Val Tyr Ser Asn Lys Thr Val Ser Cys Phe
325 330 335Ala Ile Tyr Thr Thr Ser Asp Lys Ala Ile Glu Leu Tyr Asp
Lys Ile 340 345 350Glu Lys Phe Lys Val Asp Phe Lys Ser Arg His Ala
Cys Glu Leu Gly 355 360 365Cys Ile Leu Leu Phe Ile Thr Leu Ser Lys
His Arg Val Ser Ala Ile 370 375 380Lys Asn Phe Cys Ser Thr Phe Cys
Thr Ile Ser Phe Leu Ile Cys Lys385 390 395 400Gly Val Asn Lys Met
Pro Glu Met Tyr Asn Asn Leu Cys Lys Pro Pro 405 410 415Tyr Lys Leu
Leu Gln Glu Asn Lys Pro Leu Leu Asn Tyr Glu Phe Gln 420 425 430Glu
Lys Glu Lys Glu Ala Ser Cys Asn Trp Asn Leu Val Ala Glu Phe 435 440
445Ala Cys Glu Tyr Glu Leu Asp Asp His Phe Ile Ile Leu Ala His Tyr
450 455 460Leu Asp Phe Ala Lys Pro Phe Pro Cys Gln Lys Cys Glu Asn
Arg Ser465 470 475 480Arg Leu Lys Pro His Lys Ala His Glu Ala His
His Ser Asn Ala Lys 485 490 495Leu Phe Tyr Glu Ser Lys Ser Gln Lys
Thr Ile Cys Gln Gln Ala Ala 500 505 510Asp Thr Val Leu Ala Lys Arg
Arg Leu Glu Met Leu Glu Met Thr Arg 515 520 525Thr Glu Met Leu Cys
Lys Lys Phe Lys Lys His Leu Glu Arg Leu Arg 530 535 540Asp Leu Asp
Thr Ile Asp Leu Leu Tyr Tyr Met Gly Gly Val Ala Trp545 550 555
560Tyr Cys Cys Leu Phe Glu Glu Phe Glu Lys Lys Leu Gln Lys Ile Ile
565 570 575Gln Leu Leu Thr Glu Asn Ile Pro Lys Tyr Arg Asn Ile Trp
Phe Lys 580 585 590Gly Pro Ile Asn Ser Gly Lys Thr Ser Phe Ala Ala
Ala Leu Ile Asp 595 600 605Leu Leu Glu Gly Lys Ala Leu Asn Ile Asn
Cys Pro Ser Asp Lys Leu 610 615 620Pro Phe Glu Leu Gly Cys Ala Leu
Asp Lys Phe Met Val Val Phe Glu625 630 635 640Asp Val Lys Gly Gln
Asn Ser Leu Asn Lys Asp Leu Gln Pro Gly Gln 645 650 655Gly Ile Asn
Asn Leu Asp Asn Leu Arg Asp His Leu Asp Gly Ala Val 660 665 670Ala
Val Ser Leu Glu Lys Lys His Val Asn Lys Lys His Gln Ile Phe 675 680
685Pro Pro Cys Ile Val Thr Ala Asn Asp Tyr Phe Ile Pro Lys Thr Leu
690 695 700Ile Ala Arg Phe Ser Tyr Thr Leu His Phe Ser Pro Lys Ala
Asn Leu705 710 715 720Arg Asp Ser Leu Asp Gln Asn Met Glu Ile Arg
Lys Arg Arg Ile Leu 725 730 735Gln Ser Gly Thr Thr Leu Leu Leu Cys
Leu Ile Trp Cys Leu Pro Asp 740 745 750Thr Thr Phe Lys Pro Cys Leu
Gln Glu Glu Ile Lys Asn Trp Lys Gln 755 760 765Ile Leu Gln Ser Glu
Ile Ser Tyr Gly Lys Phe Cys Gln Met Ile Glu 770 775 780Asn Val Glu
Ala Gly Gln Asp Pro Leu Leu Asn Ile Leu Ile Glu Glu785 790 795
800Glu Gly Pro Glu Glu Thr Glu Glu Thr Gln Asp Ser Gly Thr Phe Ser
805 810 815Gln71269DNAMerkel cell Polyomavirus MCV-IC13 7tcataattct
tgtgtttggc tttctttttg agaggccttt tgaggtcctt tcagtggcgc 60cttgccctta
tcctgctgat tactttggaa tgttactgct gctggggcaa cagagggctt
120tgggtaaaca gttttctcct gcccaaattt atctaaaaat ctgacaatat
caggatcacc 180aggtaattgt tctgacccct catatattct aacctcttct
acctgattat
cttttccttc 240cataggttgg cctgacactt ttggcattaa gttgctgaag
agtgagttta ttaaattaac 300tactgggtag gggtttttca cccatctttt
tctgaaagta acattaaaat atctaggcaa 360cccatgaaga gccatttttc
cactggtttt aaacagaaac cccactatgt ctgcacagct 420aataaatagg
ccatctcctt tgcatagagg gcccactcca ttctcatcta aaaggacagt
480agttagagta ttactaaatt gaagaactgt aggagtctga gagcctgtct
gaatagaccc 540atagtatcta ctgttttcat ttttagaagg atcaggacac
catacttcta taggataatt 600tccatcttta tctaattttg ctttagcttg
tggatctagg ccctgatttt taggtgtcat 660ttttcttccc aatacagttt
caattgtaat aggcccacca tttgtagttt ttggatactc 720agtctggtaa
tctaaaacta ggccttgcaa atccagaggt tctcccccaa tggcaaacat
780atggtaattt actcctgaca caggaatacc agcaccataa tcatgaactc
ttttcatgtc 840ccaataatga acattaatta aagaacttat tccaactact
tctgttttaa cagatattgc 900ctcccacatc tgcaatgtgt cacaggtaat
atcctcattt agcattggca gagacactct 960tgccacactg taagctggca
aattttcctt gatgggctga tctggagatg atccctttgg 1020ctgcaggtca
taagtataag tataccagtt tgaagtagta ggaagatcag gggaattaac
1080tcccattctt ggattcaaat acaactcaat ttgggtaatg ctatcttctc
cagtaaccac 1140agttaatact tccactcctc ctttaacaag cagttttgga
actgaggcaa cattagggca 1200gcatcccggc ttaggtatac attgcctttt
gggtgtttta caggtggatg atgcttttct 1260ttttggtgc 12698423PRTMerkel
cell Polyomavirus MCV-IC13 8Met Ala Pro Lys Arg Lys Ala Ser Ser Thr
Cys Lys Thr Pro Lys Arg1 5 10 15Gln Cys Ile Pro Lys Pro Gly Cys Cys
Pro Asn Val Ala Ser Val Pro 20 25 30Lys Leu Leu Val Lys Gly Gly Val
Glu Val Leu Thr Val Val Thr Gly 35 40 45Glu Asp Ser Ile Thr Gln Ile
Glu Leu Tyr Leu Asn Pro Arg Met Gly 50 55 60Val Asn Ser Pro Asp Leu
Pro Thr Thr Ser Asn Trp Tyr Thr Tyr Thr65 70 75 80Tyr Asp Leu Gln
Pro Lys Gly Ser Ser Pro Asp Gln Pro Ile Lys Glu 85 90 95Asn Leu Pro
Ala Tyr Ser Val Ala Arg Val Ser Leu Pro Met Leu Asn 100 105 110Glu
Asp Ile Thr Cys Asp Thr Leu Gln Met Trp Glu Ala Ile Ser Val 115 120
125Lys Thr Glu Val Val Gly Ile Ser Ser Leu Ile Asn Val His Tyr Trp
130 135 140Asp Met Lys Arg Val His Asp Tyr Gly Ala Gly Ile Pro Val
Ser Gly145 150 155 160Val Asn Tyr His Met Phe Ala Ile Gly Gly Glu
Pro Leu Asp Leu Gln 165 170 175Gly Leu Val Leu Asp Tyr Gln Thr Glu
Tyr Pro Lys Thr Thr Asn Gly 180 185 190Gly Pro Ile Thr Ile Glu Thr
Val Leu Gly Arg Lys Met Thr Pro Lys 195 200 205Asn Gln Gly Leu Asp
Pro Gln Ala Lys Ala Lys Leu Asp Lys Asp Gly 210 215 220Asn Tyr Pro
Ile Glu Val Trp Cys Pro Asp Pro Ser Lys Asn Glu Asn225 230 235
240Ser Arg Tyr Tyr Gly Ser Ile Gln Thr Gly Ser Gln Thr Pro Thr Val
245 250 255Leu Gln Phe Ser Asn Thr Leu Thr Thr Val Leu Leu Asp Glu
Asn Gly 260 265 270Val Gly Pro Leu Cys Lys Gly Asp Gly Leu Phe Ile
Ser Cys Ala Asp 275 280 285Ile Val Gly Phe Leu Phe Lys Thr Ser Gly
Lys Met Ala Leu His Gly 290 295 300Leu Pro Arg Tyr Phe Asn Val Thr
Phe Arg Lys Arg Trp Val Lys Asn305 310 315 320Pro Tyr Pro Val Val
Asn Leu Ile Asn Ser Leu Phe Ser Asn Leu Met 325 330 335Pro Lys Val
Ser Gly Gln Pro Met Glu Gly Lys Asp Asn Gln Val Glu 340 345 350Glu
Val Arg Ile Tyr Glu Gly Ser Glu Gln Leu Pro Gly Asp Pro Asp 355 360
365Ile Val Arg Phe Leu Asp Lys Phe Gly Gln Glu Lys Thr Val Tyr Pro
370 375 380Lys Pro Ser Val Ala Pro Ala Ala Val Thr Phe Gln Ser Asn
Gln Gln385 390 395 400Asp Lys Gly Lys Ala Pro Leu Lys Gly Pro Gln
Lys Ala Ser Gln Lys 405 410 415Glu Ser Gln Thr Gln Glu Leu
4209725DNAMerkel cell Polyomavirus MCV-IC13 9tacaggtgga tgatgctttt
ctttttggtg ccatcttcaa ttacttgtaa ttcaggagaa 60atatatccac taaggcctag
taccagagga agaagccaat ctggagtttg ctgctgcaga 120gttcctccta
tatgttcagg aattaatata gcctctcctg ataaaaggcc ctgattctga
180gaagcagttg tgtgaaagac ccaccggcta tttagtatca gattcactag
gtttgattgt 240atctgcagcc tagaggtagg agataaagaa ttaaaaatat
tttgccccac agaatgcagc 300aagctatttt cccactgcag aggatctagg
ctaaaggcca taagtgcatg cctcaaaacc 360tcattactac ctacccacga
aacatccctc tttacaagtg acacttgctc gcgtgacaac 420ctcaccccca
cagttattag agagcctata ccactaacag tttggagaat gaagccataa
480gttaaacctt ggttaaccaa agaagccact aatgagaaat ttgaaaactg
ttcagctgtg 540aacccaagtt gagctaaagc ctcaatgcca gaaataccct
caattgtcat taaactggag 600atctctgctt ccaaagctgc taaagcttct
cctgtaagaa tagcttccaa agttactcct 660gtggtggcac ttagttcagt
agcaatttca ccaatattgg ccagcagtgt gatgatgccc 720cccat
72510240PRTMerkel cell Polyomavirus MCV-IC13 10Met Gly Gly Ile Ile
Thr Leu Leu Ala Asn Ile Gly Glu Ile Ala Thr1 5 10 15Glu Leu Ser Ala
Thr Thr Gly Val Thr Leu Glu Ala Ile Leu Thr Gly 20 25 30Glu Ala Leu
Ala Ala Leu Glu Ala Glu Ile Ser Ser Leu Met Thr Ile 35 40 45Glu Gly
Ile Ser Gly Ile Glu Ala Leu Ala Gln Leu Gly Phe Thr Ala 50 55 60Glu
Gln Phe Ser Asn Phe Ser Leu Val Ala Ser Leu Val Asn Gln Gly65 70 75
80Leu Thr Tyr Gly Phe Ile Leu Gln Thr Val Ser Gly Ile Gly Ser Leu
85 90 95Ile Thr Val Gly Val Arg Leu Ser Arg Glu Gln Val Ser Leu Val
Lys 100 105 110Arg Asp Val Ser Trp Val Gly Ser Asn Glu Val Leu Arg
His Ala Leu 115 120 125Met Ala Phe Ser Leu Asp Pro Leu Gln Trp Glu
Asn Ser Leu Leu His 130 135 140Ser Val Gly Gln Asn Ile Phe Asn Ser
Leu Ser Pro Thr Ser Arg Leu145 150 155 160Gln Ile Gln Ser Asn Leu
Val Asn Leu Ile Leu Asn Ser Arg Trp Val 165 170 175Phe His Thr Thr
Ala Ser Gln Asn Gln Gly Leu Leu Ser Gly Glu Ala 180 185 190Ile Leu
Ile Pro Glu His Ile Gly Gly Thr Leu Gln Gln Gln Thr Pro 195 200
205Asp Trp Leu Leu Pro Leu Val Leu Gly Leu Ser Gly Tyr Ile Ser Pro
210 215 220Glu Leu Gln Val Ile Glu Asp Gly Thr Lys Lys Lys Ser Ile
Ile His225 230 235 24011591DNAMerkel cell Polyomavirus MCV-IC13
11ttacaggtgg atgatgcttt tctttttggt gccatcttca attacttgta attcaggaga
60aatatatcca ctaaggccta gtaccagagg aagaagccaa tctggagttt gctgctgcag
120agttcctcct atatgttcag gaattaatat agcctctcct gataaaaggc
cctgattctg 180agaagcagtt gtgtgaaaga cccaccggct atttagtatc
agattcacta ggtttgattg 240tatctgcagc ctagaggtag gagataaaga
attaaaaata ttttgcccca cagaatgcag 300caagctattt tcccactgca
gaggatctag gctaaaggcc ataagtgcat gcctcaaaac 360ctcattacta
cctacccacg aaacatccct ctttacaagt gacacttgct cgcgtgacaa
420cctcaccccc acagttatta gagagcctat accactaaca gtttggagaa
tgaagccata 480agttaaacct tggttaacca aagaagccac taatgagaaa
tttgaaaact gttcagctgt 540gaacccaagt tgagctaaag cctcaatgcc
agaaataccc tcaattgtca t 59112181PRTMerkel cell Polyomavirus
MCV-IC13 12Met Thr Ile Glu Gly Ile Ser Gly Ile Glu Ala Leu Ala Gln
Leu Gly1 5 10 15Phe Thr Ala Glu Gln Phe Ser Asn Phe Ser Leu Val Ala
Ser Leu Val 20 25 30Asn Gln Gly Leu Thr Tyr Gly Phe Ile Leu Gln Thr
Val Ser Gly Ile 35 40 45Gly Ser Leu Ile Thr Val Gly Val Arg Leu Ser
Arg Glu Gln Val Ser 50 55 60Leu Val Lys Arg Asp Val Ser Trp Val Gly
Ser Asn Glu Val Leu Arg65 70 75 80His Ala Leu Met Ala Phe Ser Leu
Asp Pro Leu Gln Trp Glu Asn Ser 85 90 95Leu Leu His Ser Val Gly Gln
Asn Ile Phe Asn Ser Leu Ser Pro Thr 100 105 110Ser Arg Leu Gln Ile
Gln Ser Asn Leu Val Asn Leu Ile Leu Asn Ser 115 120 125Arg Trp Val
Phe His Thr Thr Ala Ser Gln Asn Gln Gly Leu Leu Ser 130 135 140Gly
Glu Ala Ile Leu Ile Pro Glu His Ile Gly Gly Thr Leu Gln Gln145 150
155 160Gln Thr Pro Asp Trp Leu Leu Pro Leu Val Leu Gly Leu Ser Gly
Tyr 165 170 175Ile Ser Pro Glu Leu 180
* * * * *